Erythropoietin variants

ABSTRACT

The present invention relates to novel endogenous variants of erythropoietin (EPO) and their use for treatment or prevention of a condition associated with tissue damage due to cell death (apoptosis, necrosis) and inflammation, in particular for neuroprotection, e.g. treatment of acute (for example stroke) and chronic disease (for example ALS) of the nervous system.

FIELD OF THE INVENTION

The present invention relates to novel endogenous variants oferythropoietin (EPO) and their use for treatment or prevention of acondition associated with tissue damage due to cell death (apoptosis,necrosis) and inflammation, in particular for neuroprotection, e.g.treatment of acute (for example stroke) and chronic disease (for exampleALS) of the nervous system.

BACKGROUND OF THE INVENTION

Stroke is a debilitating disease which affects more than 400,000 personsper year in the United States and is the third most common cause ofdeath in the United States. In addition one-half of neurology inpatientshave stroke related problems. At current trends, this number isprojected to jump to one million per year by the year 2050. When thedirect costs (care and treatment) and the indirect costs (lostproductivity) of strokes are considered together, strokes put a burdenof $43.3 billion per year on the society of the United States alone.About ⅓ of patients die in the first three months, ⅓ remain with severedisabilities, and only ⅓ recover with acceptable outcome. In 1990cerebrovascular diseases were the second leading cause of deathworldwide, killing over 43.3 million people world wide. Thus, from apublic health perspective, stroke is one of the most relevant diseases.

stroke is characterized by the sudden loss of circulation to an area ofthe brain, resulting in a corresponding loss of neurologic function.Also called cerebrovascular accident or stroke syndrome, stroke is anonspecific term encompassing a heterogeneous group of pathophysiologiccauses, including thrombosis, embolism, and hemorrhage. Strokescurrently are classified as either hemorrhagic or ischemic. Acuteischemic stroke refers to strokes caused by thrombosis or embolism andaccount for 80% of all strokes.

Ischemic strokes result from blockage of the arteries that supply thebrain, most commonly in the branches of the internal carotid arteries.The blockage usually results when a piece of a blood clot (thrombus) orof a fatty deposit (atheroma) due to atherosclerosis breaks off(becoming an embolus), travels through the bloodstream, and lodges in anartery that supplies the brain. Blood clots may form when a fattydeposit in the wall of an artery ruptures. The rupture of such a fattydeposit may also form when a large fatty deposit slows blood flow,reducing it to a trickle. Blood that flows slowly is more likely toclot. Thus, the risk of a clot forming in and blocking a narrowed arteryis high. Blood clots may also form in other areas, such as in the heartor on a heart valve. Strokes due to such blood clots are most commonamong people who have recently had heart surgery and people who have aheart valve disorder or an abnormal heart rhythm (arrhythmia),especially atrial fibrillation. Also, in certain disorders such as anexcess of red blood cells (polycythemia), the risk of blood clots isincreased because the blood is thickened.

As ischemic stroke can also result, if the blood flow to the brain isreduced, as may occur when a person loses a lot of blood or has very lowblood pressure. Occasionally, an ischemic stroke occurs when blood flowto the brain is normal but the blood does not contain enough oxygen.Disorders that reduce the oxygen content of blood include severe anemia(a deficiency of red blood cells), suffocation, and carbon monoxidepoisoning. Usually, brain damage in such cases is widespread (diffuse),and coma results. An ischemic stroke can occur, if inflammation orinfection narrows blood vessels that supply the brain, Similarly, drugssuch as cocaine and amphetamines can cause spasm of the arteries, whichcan lead to a narrowing of the arteries supplying the brain to such anextent that a stroke is caused.

The brain requires glucose and oxygen to maintain neuronal metabolismand function. The inadequate delivery of oxygen to the brain leads to ahypoxia and ischemia results from insufficient cerebral blood flow. Theconsequences of cerebral ischemia depend on the degree and the durationof reduced cerebral blood flow. Neutrons can tolerate ischemia for 30-60minutes. If flow is not re-established to the ischemia area, a series ofmetabolic processes ensue. The neurons become depleted of ATP and switchover to anaerobic glycolysis, a much less efficient pathway. Lactateaccumulates and the intracellular pH decreases. Without an adequatesupply of ATP, ion pumps in the plasma membrane fail. The resultinginflux of sodium, water, and calcium into the cell causes rapid swellingof neurons and glial cells. Membrane depolarization also stimulates themassive release of the amino acids glutamate and aspartate, both ofwhich act as excitatory neurotransmitters in the brain. Glutamatefurther activates sodium and calcium ion channels in the neuronal cellmembrane namely the well characterized N-methyl-D-aspartate (NMDA)calcium channel. Excessive calcium influx causes the disorderedactivation of a wide range of enzyme systems (proteases, lipases, andnucleases). These enzymes and their metabolic products, such as oxygenfree radicals, damage cell membranes, genetic material, and structuralproteins in the neurons, ultimately leading to the cell death of neurons(Dimagl, U. et al. (1999) Trends Neurosci. 22: 391-397).

Strokes begin suddenly, develop rapidly, and cause death of brain tissuewithin minutes to days. In the ischemic brain, we commonly distinguishtwo tissue volumes—the core of the infarction and the surrounding zone,known as ischemic penumbra—the underperfused and metabolicallycompromised margin surrounding the irrevocably damaged core. Core andpenumbra are characterized by two different kinds of cell death;necrosis and apoptosis (which is also called programmed cell death ordelayed neuronal cell death). The severe perfusion deficit in the corecauses a breakdown of metabolic processes, cellular energy supply andion homeostasis, which causes the cells to lose their integrity withinminutes. Thus, acute necrosis of cell and tissue prevails in the core.In the penumbra, some residual perfusion is maintained by collateralvessels, which may be unable to maintain the full functional metabolism,but prevents immediate structural disintegration. However, over time(hours to several days), the alteration of cellular homeostasis causesmore and more cells to die, and the volume of the infarction increases.The penumbra has thus to be considered as tissue at risk during thematuration of the infarct. In this region, apoptosis and inflammatorysignaling cascades play an important role. It may initially constitute50% of the volume that will end up as infarction. The mechanisms thatlead to delayed cell death provide targets for a specificneuroprotective therapy in brain regions challenged by ischemia, butwhich are still viable.

Therapeutic options so far are highly disappointing; Thrombolysis withrtPA, the only therapy with proven efficacy in a major clinical trial(NINDS), is only effective within a three hour time window, limiting itsapplication to only a few percent of patients wish ischemic stroke. Inother words, besides basic supportive therapy, at present more than 95%of strokes cannot be treated specifically. This is in sharp contrast toour knowledge concerning the basic pathophysiology of this disease,which has emerged over the last decade. In particular, extensiveknowledge has accumulated on mechanisms of parenchymal brain damage andendogenous neuroprotection, as well as functional and structuralreorganization.

Recently, attention has focused on potential therapeutic roles forendogenous brain proteins possessing neuroprotective properties. EPO, aglycoprotein hormone produced primarily by cells of the peritubularcapillary endothelium of the kidney, which is a member of the growthhormone/prolacton cytokine family (Zhu Y. and D'Andrea A. D: (1994)Curr. Opin. Hematol. 1: 113-118) is a promising candidate. Although EPOwas first characterized and is now widely known for its role as ahaematopoietic hormone the detection of EPO and its receptor (EPOR) inrodent and human brain tissue as well as in cultured neurons andastrocytes expanded the search for other biological roles of EPO.

In the brain, a paracrine EPO/(Epo-R)₂ system exists independent of theendocrine system of adult erythropoiesis; neurones express (Epo-R)₂ andastrocytes produce EPO (Ruscher et al. (2002) J. Neurosci. 22,10291-301; Prass et al. (2003) Stroke 34,1981-1986). It was demonstratedin vivo and in vivo that EPO is a potent inhibitor of neuronal apoptosisinduced by ischemia and hypoxia (Ruscher et al. (2002) J. Neurosci. 22,10291-301; Bernaudin, M., et al. (1999) J Cereb Blood Flow Metab.19:643-51; Morishita, E., et al. (1997) Neuroscience. 76: 105-16). Itwas reported by several groups that addition of EPO to neuronal culturesprotects against hypoxic and glutamic acid toxicity (Henn F. A: andBraus D.F. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249: 48-56,Vogeley K. et al. (2000) Am. J. Psychiatry 157: 34-39) and reducesneurologic dysfunction in rodent models of strike (Brines M. L. et al.(2000) Proc. Natl. Acad. Sci. U.S.A. 97: 10526-10531 and Bernaudin etal. (1999) J. Cereb. Blood Flow Metab. 10: 643-651). The promisingresults of these experiments have been corroborated in human studieswherein it was shown that EPO therapy for acute stroke is safe and mightbe beneficial (Ehrenreich H. et al. (2002) Mol. Medicine 8: 495-505) andWO 00/35475 A2. These cell and more particular neuroprotectiveproperties of EPO have led to further research in this area tosubstantiate these findings in larger trial and the use of EPO is nowproposed in other indication as well including, for example,schizophrenia (Ehrenreich H. et al. (2004) Molecular Psychiatry 9: 42-54and WO 02/20031 A2).

For the application of EPO to prevent tissue damage the hematopoieticactivity is often not required and might be detrimental if large amountsof EPO are administered to treat or ameliorate the effects of, hypoxiaor ischemia induced tissue damage. Therefore, attempts have been made tocreate EPO variants, which only exhibit the cell protective property butnot the hematopoietic properties. U.S. 2003/0130197 describes peptidemimetics of EPO for the treatment of neurodegenerative disorders, whichbear no sequence homology to naturally occuring EPO or fragmentsthereof. U.S. Pat. No. 6,531,121 discloses a asialoerythropoietin whichis generated by complete desialylation of recombinant EPO showed anincreased ability to cross the endothelial cell barrier and had adecreased hematopoietic activity. Carbamylated erythropoietin (CEPO) wasalso shown to exhibit a tissue protective effect but no erythropoieticeffect (Leist et al. (2004) Science 305: 239-242 and WO 2005/025696 A1.

Finally, it was shown that a 17-mer peptide of EPO inhibited cell deathof two neuronal cell lines, SK-N-MC and NS20Y (Campana W. M. et al.(1998) Int. J. Mol. Medicine 1: 235-241), while at the same time havingno hematopoietic activity. However, 1 ng/ml of the EPO peptide wasneeded to elicit the same antiapoptotic effect as 100 pg/ml recombinantEPO (rhEPO) in NS20Y cells and as 400 pg/ml rhEPO in SK-N-MC cells.Given the apparent molecular weight of rhEPO of about 66,000 g/mol (thecalculated molecular weight is about 33,000 g/mol but does not includethe weight of oligosaccharide residues comprised in rhEPO) and of about1.900 g/mol of the EPO peptide a concentration of 1.52 pmol/l and 6.6pmol/l, respectively, of rhEPO and 1 nmol/l of the EPO peptide elicitedthe same level of a cell protective effect. Consequently, the EPOpeptide is between 650-fold to 165-fold less active than rhEPO inprevention of cell death. It is evident from this figures that the EPOregion comprised in the 17-mer does not play a major role in the cellprotective function of EPO. Therefore, all EPO variants, which have adecreased hematopoietic activity known in the prior art suffer from thedisadvantage that they are not natural occurring since they have eitherlost their natural glycosylation or they are artificial truncationsand/or they have vastly diminished cell protective activity, if comparedto rhEPO. Therefore, there is a need in the prior art to provide an EPOderivative, which is close to the naturally occurring EPO and which hasthe same or better tissue protecting activity as rhEPO but less or nohematopoietic, in particular erythropoietic activity.

This problem is solved by the provision of new EPO variants, which werefound to occur naturally in human and mouse tissue (brain, kidney) andwhich exhibit a cell protective activity similar or better to rhEPO butwhich do not exhibit any significant hematopoietic activity.

SUMMARY OF THE INVENTION

In one aspect the present invention is concerned with an EPO variantencoding polynucleotide selected from the group consisting of:

-   -   (a) polynucleotides encoding at least the mature form of the        polypeptides termed hs3, h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3,        mG5, m301 and mK3 having the deduced amino acid sequence as        shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22,        respectively;    -   (b) polynucleotides having the coding sequence, as shown in SEQ        ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 encoding at        least the mature form of the polypeptide;    -   (c) polynucleotide encoding a humanized version of the        polypeptides mS, mG3, mG5, M301 and mK3 having the deduced amino        acid sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22,    -   (d) polynucleotides encoding a polypeptide comprising a fusion        of an amino acid sequence selected from the group of amino acid        sequences as shown in SEQ ID NO 24, 26, 28, and 30, at the        N-terminus of an amino acid sequence selected from the group of        amino acid sequences as shown in SEQ ID NO 32, 34, 36, and 38;    -   (e) polynucleotides comprising a fusion of polynucleotide        sequences selected from the group of polynucleotide sequences as        shown in SEQ ID NO 23, 25, 27, and 29, 5′ of a polynucleotide        sequence selected from the group of polynucleotide sequences as        shown in SEQ ID NO 31, 33, 35, and 37;    -   (f) polynucleotides encoding a derivative of a polypeptide        encoded by a polynucleotide of any one of (a) to (e), wherein in        said derivative between 1 and 10 amino acid residues are        conservatively substituted compared to said polypeptide, and        said derivative has cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity;    -   (g) polynucleotides encoding a fragment of a polypeptide encoded        by a polynucleotide of any one of (a) to (f), wherein in said        fragment between 1 and 10 amino acid residues are N- and/or        C-terminally deleted and/or between 1 and 10 amino acids are        deleted N- and or C-terminally of the junction compared to said        polypeptide, and said fragment has cell protective and in        particular neuroprotective activity but essentially no        hematopoietic activity;    -   (h) polynucleotides which are at least 50% identical to a        polynucleotide as defined in any one of (a) to (g) and which        code for a polypeptide having cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity; and    -   (i) polynucleotides the complementary strand of which hybridizes        under stringent conditions to a polynucleotide as defined in any        one of (a) to (h) and which code for a polypeptide having cell        protective and in particular neuroprotective activity but        essentially no hematopoietic activity;        or the complementary strand of such a polynucleotide.

A further aspect of the present invention is a homolog of anerythropoietin (EPO) variant encoding polynucleotide from another highereukaryotic species.

A further aspect of the present invention is an EPO variant encodingpolynucleotide selected from the group consisting of:

-   -   (a) polynucleotides encoding an EPO variant polypeptide, which        comprises the N-terminal part of full length EPO including helix        A and which lacks at least one of the following:        -   (i) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids between            helix A and helix B;        -   (ii) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,            27 or 28 amino acids of helix B;    -   (iii) a fragment of at least 2 amino acids, preferably 3, 4, 5,        or 6 amino acids between helix B and helix C;        -   (iv) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino            acids of helix C;        -   (v) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,            or 27 amino acids between helix C and D; and/or        -   (vi) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 of helix D;        -   wherein said variant has cell protective and in particular            neuroprotective activity but essentially no hematopoietic            activity.    -   (b) polynucleotides encoding a derivative of a polypeptide        encoded by a polynucleotide of any one of (a), wherein in said        derivative between 1 and 10 amino acid residues are        conservatively substituted compared to said polypeptide, and        said derivative has cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity;    -   (c) polynucleotides the complementary strand of which hybridizes        under stringent conditions to a polynucleotide as defined in any        one of (a) to (b) and which code for a polypeptide having cell        protective and in particular neuroprotective activity but        essentially no hematopoietic activity;        or the complementary strand of such a polynucleotide.

In a preferred aspect the polynucleotide of the present invention whichis DNA, genomic DNA or RNA.

In another aspect the preset invention is concerned with a vectorcontaining the polynucleotide of the present invention. It is preferredthat the polynucleotide contained in the vector is operatively linked toexpression control sequences allowing expression in prokaryotic and/oreukaryotic host cells.

Another aspect of the invention is a host cell genetically engineeredwith the polynucleotide of the present invention or the vector of thepresent invention.

Another aspect of the invention is a transgenic non-human animalcontaining a polynucleotide of the present invention, a vector of thepresent invention and/or a host cell of the present invention.

Another aspect of the invention is a process for producing an EPOvariant polypeptide encoded by the polynucleotide of the presentinvention comprising: culturing the host cell of the present inventionand recovering the polypeptide encoded by said polynucleotide.

In a preferred embodiment the process of the present invention, furthercomprises the step of modifying said EPO variant, wherein themodification is selected from the group consisting of oxidation,sulfation, phosphorylation, addition of oligosaccharides or combinationsthereof.

Another aspect of the invention is a process for producing cells capableof expressing at least one of the EPO variants comprising geneticallyengineering cells in vitro with the vector of the invention, whereinsaid EPO variant polypeptide(s) is(are) encoded by a polynucleotide ofthe invention.

Another aspect of the invention is a polypeptide having the amino acidsequence encoded by a polynucleotide of the present invention orobtainable by the process of the present invention.

Another aspect of the invention is an antibody specifically binding tothe polypeptide of the present invention.

Another aspect of the invention is a pharmaceutical compositioncomprising a polynucleotide of the present invention, a vector of thepresent invention, a host cell of the present invention, a polypeptideof the present invention and/or an antibody of the present invention anda one or more pharmaceutically acceptable carrier.

Another aspect of the invention is the use of a polynucleotide of thepresent invention, a vector of the present invention, a host cell of thepresent invention, a polypeptide of the present invention for themanufacture of a medicament for the treatment or prevention of acondition associated with tissue damage due to cell death, e.g.apoptosis and necrosis as well as by inflammation.

In a preferred use of the present invention cell death is induced byischemia, hypoxia, bacterial infection, viral infection,autoimmunologically, traumatically, chemically (e.g. metabolically,toxically) induced, or radiation induced.

In a preferred use of the present invention the condition is an acuteneurodegenerative/neuroinflammatory disorder as a chronicneurodegenerative/neuroinflammatory disorder, is an acute or chronicdisorder of the heart (e.g., myocardial infarction), lung (e.g. asthma,chronic obstructive lung disease), kidney (e.g. glomerulonephritis),liver (e.g. chronic liver failure) or pancreas (e.g. pancreatitis) orsaid condition is associated with an organ (e.g. kidney or liver) orcell transplantation (e.g. stem cell).

Preferably the acute neurodegenerative and/or neuroinflammatory disorderis selected from the group consisting of cerebral ischemia or infarctionincluding embolic occlusion and thrombotic occlusion, reperfusionfollowing acute ischemia, perinatal hypoxic-ischemic injury, cardiacarrest, intracranial hemorrhage, subarachnoidal hemorrhage andintracranial lesions (e.g. CNS trauma), spinal cord lesions,intravertebral lesions, whiplash shaken infant syndrome, infectiousencephalitis (e.g. herpes encephalitis), meningitis (e.g. bacterial),headache (e.g. migraine).

Preferably the chronic neurodegenerative/neuroinflammatory disorder isselected from the group consisting of dementias (e.g. Alzheimer'sdisease, vascular dementias), Pick's disease, diffuse Lewy body disease,progressive supranuclear palsy (Steel-Richardson syndrome), multiplesclerosis, multiple system atrophy (including Shy-Drager syndrome),chronic epileptic conditions associated with neurodegeneration, motorneuron diseases, degenerative ataxias, cortical basal degeneration,ALS-Parkinson's Dementia complex of Guam, subacute sclerosingpanencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies, primary progressive aphasia, striatonigraldegeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 andolivopontocerebellar degenerations, Gilles De La Tourette's disease,bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy(Kennedy's disease), primary lateral sclerosis, familial spasticparaplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease,Tay-Sach's disease, Sandhoff disease, familial spastic disease, spasticparaparesis, progressive multifocal leukoencephalopathy, familialdysautonomia (Riley-Day syndrome), polyneuropathies (e.g. diabetic,alcohol-toxic, Guillain-Barré-Syndrome, chronic inflammatorydemyelinating polyneuropathy), prion diseases, addiction, affectivedisorders (e.g. depression), schizophrenic disorders, chronic fatiquesyndrome, chronic pain (e.g. lower back pain).

In a preferred use of the present invention the condition is aging.

In a preferred use of the present invention the medicament isadministered prior to or after the onset of said condition.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

The present invention is based on the surprising observation that EPOvariants are expressed in neuronal tissue and the determination that thevariants protected neurons from damage induced by oxygen and glucosedeprivation but did not show hematopoietic activity. This behavior makesthem suitable for use as therapeutics in situations where thehematopoietic function of EPO is not required or deleterious.Accordingly a first aspect of the present invention is an EPO variantencoding polynucleotide selected from the group consisting of:

-   -   (a) polynucleotides encoding at least the mature form of the        polypeptides termed hs3, h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3,        mG5, m301 and mK3 having the deduced amino acid sequence as        shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22,        respectively;    -   (b) polynucleotides having the coding sequence, as shown in SEQ        ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 encoding at        least the mature form of the polypeptide;    -   (c) polynucleotide encoding a humanized version of the        polypeptides mS, mG3, mG5, m301 and mK3 having the deduced amino        acid sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22,    -   (d) polynucleotides encoding a polypeptide comprising a fusion        of an amino acid sequence selected from the group of amino acid        sequences as shown in SEQ ID NO 24, 26, 28, and 30, at the        N-terminus, preferably directly, i.e. without any intervening        amino acids, of an amino acid sequence selected from the group        of amino acid sequences as shown in SEQ ID NO 32, 34, 36, and        38;    -   (e) polynucleotides comprising a fusion of polynucleotide        sequences selected from the group of polynucleotide sequences as        shown in SEQ ID NO 23, 25, 27, and 29, 5′, preferably directly        5′, i.e. without any intervening polynucleotides, of a        polynucleotide sequence selected from the group of        polynucleotide sequences as shown in SEQ ID NO 31, 33, 35, and        37;    -   (f) polynucleotides encoding a derivative of a polypeptide        encoded by a polynucleotide of any one of (a) to (e), wherein in        said derivative between 1 and 10 amino acid residues are        conservatively substituted compared to said polypeptide, and        said derivative has cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity;    -   (g) polynucleotides encoding a fragment of a polypeptide encoded        by a polynucleotide of any one of (a) to (f), wherein in said        fragment between 1 and 10 amino acid residues are N- and/or        C-terminally deleted and/or between 1 and 10 amino acids are        deleted N- and or C-terminally of the junction compared to said        polypeptide, and said fragment has cell protective and in        particular neuroprotective activity but essentially no        hematopoietic activity;    -   (h) polynucleotides which are at least 50% identical to a        polynucleotide as defined in any one of (a) to (g) and which        code for a polypeptide having cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity; and    -   (i) polynucleotides the complementary strand of which hybridizes        under stringent conditions to a polynucleotide as defined in any        one of (a) to (h) and which code for a polypeptide having cell        protective and in particular neuroprotective activity but        essentially no hematopoietic activity;        or the complementary strand of such a polynucleotide.

The invention further relates to a peptide of an EPO variant encodingpolynucleotide selected from the group consisting of:

-   -   (a) polynucleotides encoding the polypeptides termed ha, hAma,        hAmE, hA-20 and ha-transport sequence, having the deduced amino        acid sequence as shown in SEQ ID NOs 50, 51, 52, 53 and 60        respectively;    -   (b) polynucleotides having the coding sequence, as shown in SEQ        ID NOs: 55, 56, 57, 58 and 61 encoding at least the mature form        of the polypeptide;    -   (c) polynucleotides encoding a derivative of a polypeptide        encoded by a polynucleotide of any one of (a) to (b), wherein in        said derivative between 1 and 10 amino acid residues are        conservatively substituted compared to said polypeptide, and        said derivative has cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity;    -   (d) polynucleotides encoding a fragment of a polypeptide encoded        by a polynucleotide of any one of (a) to (b), wherein in said        fragment between 1 and 10 amino acid residues are N- and/or        C-terminally deleted and/or between 1 and 10 amino acids are        deleted N- and or C-terminally of the junction compared to said        polypeptide, and said fragment has cell protective and in        particular neuroprotective activity but essentially no        hematopoietic activity;    -   (e) polynucleotides which are at least 50% identical to a        polynucleotide as defined to any one of (a) to (b) and which        code for a polypeptide having cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity; and    -   (i) polynucleotides the complementary strand of which hybridizes        under stringent conditions to a polynucleotide as defined in any        one of (a) to (h) and which code for a polypeptide having cell        protective and in particular neuroprotective activity but        essentially no hematopoietic activity;        or the complementary strand of such a polynucleotide.

In a further aspect the polynucleotides of the present inventioncomprise homologs of the EPO variants of the present invention derivedfrom another higher eukaryotic species, in particular from mammals, morepreferably from non-human primates; from rodents, e.g. rat, or guineapig; ruminant, e.g. cow; or sheep; horse; pig; rabbit; dog; or cat,which have cell protective and in particular neuroprotective activitybut essentially no hematopoietic activity. In this contest the termhomolog refers to a polynucleotide encoding a EPO variant derived fromanother species, which comprises essentially the same deletion as thepolynucleotides according to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 55, 56, 57, 58 or 61. A deletion of a polynucleotide isconsidered to be essentially the same, if it involves the deletion ofpolynucleotides encoding a polypeptides, which is homologous to therespectively deleted polypeptides in the EPO variant polypeptidesaccording to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 18, 20, 22 50, 51, 52, 53or 60. The criterions for determining homology between two peptidesequences are well established. For this purpose programs as BLASTP canbe used. A deletion is still considered to be essentially the same if itinvolves 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 55, 56, 57, 58 or 61more or less nucleotides as the respective deletion in SEQ ID NO 1, 3,5, 7, 9, 11, 13, 15, 17, 19, or 21, which are also depicted in FIG. 1and 2.

A further aspect of the present invention is an EPO variant encodingpolynucleotide selected from the group consisting of:

-   -   (a) polynucleotides encoding an EPO variant polypeptide, which        comprises the N-terminal part of full length EPO including helix        A and which lacks at least one of the following:        -   (i) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids between            helix A and helix B;        -   (ii) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26,            27 or 28 amino acids of helix B;    -   (iii) a fragment of at least 2 amino acids, preferably 3, 4, 5,        or 6 amino acids between helix B and helix C;        -   (iv) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 amino acids            of helix C;        -   (v) a fragment of at least 10 amino acids, preferably 11,            12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,            or 27 amino acids between helix C and D; and/or    -   (vi) a fragement of at least 10 amino acids, preferably 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 of helix D;        -   wherein, said variant has cell protective and in particular            neuroprotective activity but essentially no hematopoietic            activity.    -   (b) polynucleotides encoding a derivative of a polypeptide        encoded by a polynucleotide of any one of (a), wherein in said        derivative between 1 and 10 amino acid residues are        conservatively substituted compared to said polypeptide, and        said derivative has cell protective and in particular        neuroprotective activity but essentially no hematopoietic        activity;    -   (c) polynucleotides the complementary strand of which hybridizes        under stringent conditions to a polynucleotide as defined in any        one of (a) to (b) and which code for a polypeptide having cell        protective and in particular neuroprotective activity but        essentially no hematopoietic activity; or the complementary        strand of such a polynucleotide.

In this context helix A, B, C, and D of the EPO polypeptide are regionshomologous to the respective helix A, B, C, and D regions of full lengthEPO from mouse and human as outlined in FIG. 4. It is well known in theart how to determine homologies between two polypeptide sequences andsomeone of skill in the art will be capable to align a given EPOpolypeptide sequence derived, e.g. from another species, and todetermine the respective position of helix A, B, C, and D in this EPOpolypeptide. It is preferred that the EPO variant polynucleotide isderived from a higher eukaryote, in particular a mammal or bird.Preferred mammals are humans, non-human primates; rodents, e.g. rat, orguinea pig; ruminant, e.g. cow; or sheep; horse; pig; rabbit; dog; orcat. A larger member of such full length EPO encoding polynucleotidesfrom various species are known, including without limitation cat (GeneHank Acc. L10606), pig (Gene Bank Acc. 10607), sheep (Gene Bank Acc.10610), dog (Gene Bank Acc. L13027), macaque (Gene Bank Acc. M18189),rhesus monkey (Gene Bank Acc. L10609), mouse (Gene Bank Acc. 12930), rat(Gene Bank Acc. L10608), human (Gene Bank Acc. M11319) Bos taurus (GeneBank Acc. U44762) and Bos indicus (Gene Bank Acc. L41354).

Preferably the polynucleotides encoding an EPO variant polypeptide lacksthe following: (i); (ii): (iii); (iv); (v); (vi); (i) and (ii); (i) and(iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (iii); (ii) and(iv); (ii) and (v); (ii) and (vi); (iii) and (iv); (iii) and (v); (iii)and (vi); (iv) and (v); (iv) and (vi); (v) and (vi); (i), (ii) and(iii); (i), (ii) and (iv), (i), (ii) and (v), (i), (ii), (vi), (i),(iii) and (iv); (i), (iii) and (v); (i), (iii) and (vi); (i), (iv) and(v); (i), (iv) and (vi); (i), (v) and (vi); (ii), (iii) and (iv); (ii),(iii) and (v); (ii), (iii) and (vi); (ii), (iv) and (v); (ii), (iv) and(vi); (ii), (v) and (vi); (iii), (iv) and (v); (iii), (iv) and (vi);(iii), (v) and (vi); or (iv), (v) and (vi).

A polypeptide that exhibits cell protective activity is a polypeptidethat has at least 50% (e.g., at least: 55%; 60%; 65%; 70%; 75%; 80%;85%; 90%; 95%; 99%; 99.5%; or 100% or even more) of the ability of therespective EPO variant to protect neurons from damage by apoptosis,wherein the apoptosis is induced by oxygen or glucose deprivation, bychemical or radiation exposure or by viral or bacteria infection. Assaysto determine damage to cells, in particular to neuronal cells are knownin the art. A suitable assays is the oxygen glucose deprivation assaydescribed herein below. In the described assay the readout is the amountof lactate dehydrogenase activity (LDH). However, a variety of othermethods exist which allow assessing the damage induced in a cell and inparticular the amount of cell death (e.g. apoptosis, necoris). Theseassays include without limitation Tunnel assays, MTT-assay, life/deathassay by staining (e.g. Ethidium bromide and acridine orange staining),caspase assay, electron microscopy, DNA-laddering, which are all wellknown in the art.

An EPO variant polypeptide that exhibits essentially no hematopoieticactivity a polypeptide, which elicits in art known colony formationassays, an example of which is described below, at the same molarconcentration as the rhEPO and wt mEPO, respectively, less than 10% ofthe CFU-E (Colony forming unit-erythroblast), preferably less than 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. The respective CFU-E numbers arecalculated for a given rhEPO, wt mEPO or EPO variant by subtracting fromeach value the number of CFU-E observed in a control reaction (withoutwt or EPO variant).

In the context of the polypeptides of the present invention the term“junction” refers to the site wherein two amino acids follow each otherwhich are not consecutive in the rhEPO or mouse wt EPO and which arepotentially the result of splice events or other rearrangements in theEPO mRNA. The respective junction of the EPO variants of the presentinvention can be derived from FIG. 4, e.g. is ENIT|RVGQ for hS3,VGQQ|ALLV for h1-4, VNFY|ALLV for h1-5, KRME|PWEP for hS4, ITVP|GPVG forh1-1, LNEN|NHC for h2-1, KRME|KELM for mS, LLAN|FLRG for mG3, DTFC|RRGDfor mG5, KVNF|LRGK for m301 or LSEA|VHGR for mK3.

The polynucleotide molecules of the invention can be synthesized invitro (for example, by phosphoramidite-based synthesis) or can beobtained from a cell, such as the cell of a mammal.

The EPO variants termed mS, mG3, mG5, m301 and mK3 having the deducedamino acid sequence as shown in SEQ ID NOs 14, 16 18, 20, and 22,respectively were isolated from mouse The mouse sequence is highlyhomologous to the human sequence. An alignment of the amino acidsequences of EPO derived from humans and mouse is provided in FIG. 4. Asis apparent the mouse sequence is distinguished from the human sequenceby the lack of an alanine residue at position 8 and by the following 39substitutions (the numbering is according to the respective amino acidposition in the human EPO, the first amino acid indicated is the humanamino acid at that position and the second is the corresponding mouseamino acid): ⁴H→⁴P, ⁶C→⁶R, ⁹W→⁹T; ¹¹W→¹¹L, ¹⁸S→¹⁸L; ¹⁹L→¹⁹I; ²⁷G→²⁷C;⁴³L→⁴³I; ⁵²I→⁵²V; ⁴³T→⁵⁴M; ⁶⁰H→⁶⁰G; ⁶¹C→⁶¹P; ⁶²S→⁶²R; ⁶⁴N→⁶⁴S; ⁸⁴G→⁸⁴E;⁸⁵Q→⁸⁵E; ⁹⁵A→⁹⁵S; ¹⁰¹V→¹⁰¹I; ¹⁰³R→¹⁰³Q; ¹⁰⁴G→¹⁰⁴A; ¹⁰⁹V→¹⁰⁹A; ¹¹⁵W→¹¹⁵P;¹¹⁷P→¹¹⁷T; ¹²²V→¹²²I; ¹²⁶V→¹²⁶I; ¹³⁴T→¹³⁴S; ¹³⁸A→¹³⁸V; ¹⁴⁵A→¹⁴⁵L;¹⁴⁶I→¹⁴⁶M; ¹⁵¹A→¹⁵¹T; ¹⁵²A→¹⁵²T; ¹⁵³S→¹⁵³P; ¹⁵⁴A→¹⁵⁴P; ¹⁶⁰I→¹⁶⁰L;¹⁶²A→¹⁶²V; ¹⁶⁶R→¹⁶⁶C; ¹⁷³S→¹⁷³A; ¹⁸⁷A→¹⁸⁷V and ¹⁹⁰T→¹⁹⁰R. A humanizedmS, mG3, mG5, m301 or mK3 carries the additional alanine residue atposition 8 and/or at one or more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36 or 37 positions the human rather than themouse amino acid sequence. It is particularly preferred that mS, mG3,mG5, m301 and mK3 are fully humanized, i.e. that every amino acid at theabove outlined positions, in as far as they are present in therespective variant, is of the human sequence rather than the mousesequence. It is expected that the humanization of the mouse variantswill diminish any immunological problems, which might be encounteredwhen using in the treatment of humans.

The EPO variant nucleic acid molecules of the invention can be DNA,cDNA, genomic DNA, synthetic DNA, or, RNA, and can be double-stranded orsingle-stranded, the sense and/or an antisense strand. These moleculescan be produced by, for example, polymerase chain reaction (PCR) orgenerated by treatment with one or more restriction endonucleases. Aribonucleic acid (RNA) molecule can be produced by in vitrotranscription.

The polynucleotide molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide, i.e. the polypeptides with SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, and 22. In addition, these nucleic acid moleculesare not limited to coding sequences, e.g., they can include some or allof the non-coding sequences that lie upstream or downstream from acoding sequence.

In addition, the isolated nucleic acid molecules of the invention canencompass segments that are not found as such in the natural state.Thus, the invention encompasses recombinant nucleic acid moleculesincorporated into a vector (for example, a plasmid or viral vector) orinto the genome of a heterologous cell (or the genome of a homologouscell, at a position other than the natural chromosomal location).Recombinant nucleic acid molecules and uses therefore are discussedfurther below.

In preferred embodiments the polynucleotides of the present inventionalso comprise nucleic acid molecules which are at least 50% , preferably55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to: (a) a nucleic acidmolecule that encodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 50, 51, 52, 53 or 60 and (b) the nucleotide sequenceof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 55, 56, 57, 58 or 61respectively and which at the same time cell protective and inparticular neuroprotective activity but essentially no hematopoieticactivity

The determination of percent identity between two sequences inaccomplished using the mathematical algorithm of Karlin and Altschul(1993) Proc. Natl. Acad. Sci. U.S.A 90: 5873-5877. Such an algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et al.(1990) J. Mol. Biol. 215: 403-410. BLAST nucleotide searches areperformed with the BLASTN program, score=100, word length=12, to obtainnucleotide sequences homologous to the EPO variant polypeptide encodingnucleic acids. BLAST protein searches are performed with the BLASTPprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to the EPO variant polypeptide, respectively. To obtaingapped alignments for comparative purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs are used.

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A nucleic acid sequence encoding any of the EPOvariants disclosed herein, or a derivative or fragment thereof, can beused as a hybridization probe according to standard hybridizationtechniques. The hybridization of an EPO variant probe to DNA or RNA froma test source (e.g., a mammalian cell) is an indication of the presenceof the relevant EPO DNA or RNA in the test source. Hybridizationconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y.,6.3.1-6.3.6, 1991. Stringent conditions are defined as equivalent tohybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C.,followed by a wash in 0.2 X SSC, 0.1 % SDS at 65° C. When selecting aprobe specific for a variant carrying an internal deletion it ispreferred that the probe used to detect homologous nucleic acidsoverlaps the boundaries of the deletion, e g. hs3, h1-4, h1-5, hS4, mS,mG3, mG5 or m301. In cases where the splicing leads to an alternateC-terminus of the protein, e.g. h1-1, h2-1 or mK3 it is preferred thatthe probe used to detect homologous DNA sequences overlaps theboundaries between the known EPO sequence and the alternate C-terminus.For example, a probe could be designed, which comprises 10 complementarybases 5′ of the slice site and 10 complementary bases 3′ of the splicesite.

An “isolated DNA” is either (1) a DNA that contains sequence notidentical to that of any naturally occurring sequence, or (2), in thecontext of a DNA with a naturally-occurring sequence (e.g., a cDNA orgenomic DNA), a DNA free of at least one of the genes that flank thegene containing the DNA of interest in the genome of the organism inwhich the gene containing the DNA of interest naturally occurs. The termtherefore includes a recombinant DNA incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote. The term also includes a separate molecule suchas a cDNA where the corresponding genomic DNA has introns and thereforea different sequence; a genomic fragment that lacks at least one of theflanking genes; a fragment of DNA or genomic DNA produced by polymerasechain reaction (PCR) and that lacks at least one of the flanking genes;a restriction fragment that lacks at least one of the flanking genes; aDNA encoding a non-naturally occurring protein such as a fusion protein,mutein, or fragment of a given protein; and a nucleic acid which is adegenerate variant of a cDNA or a naturally occurring nucleic acid. Inaddition, it includes a recombinant nucleotide sequence that is part ofa hybrid gene, i.e., a gene encoding a non-naturally occurring fusionprotein. It will be apparent from the foregoing that isolated DNA doesnot mean a DNA present among hundreds to millions of other DNA moleculeswithin, for example, cDNA or genomic DNA libraries or genomic DNArestriction digests in, for example, a restriction digest reactionmixture or an electrophoretic gel slice.

A further aspect of the present invention is a vector containing thepolynucleotide(s) of the present invention or a protein encoded by apolynucleotide of the present invention. The term “vector” refers to aprotein or a polynucleotide or a mixture thereof which is capable ofbeing introduced or of introducing the proteins and/or nucleic acidcomprised into a cell. It is preferred that the proteins encoded by theintroduced polynucleotide are expressed within the cell uponintroduction of the vector.

In a preferred embodiment the vector of the present invention comprisesplasmids, phagemids, phages, cosmids, artificial mammalian chromosomes,knock-out or knock-in constructs, viruses, in particular adenoviruses,vaccinia viruses, attenuated vaccinia viruses, canary pox viruses,lentivirus (Chang, L. J. and Gay, E. E. (20001) Currr. Gene Therap.1:237-251), herpes viruses, in particular Herpes simplex virus (HSV-1Carlezon, W. A. et al. (2000) Crit. Rev. Neurobiol), baculovirus,retrovirus, adeno-associated-virus (AAV, Carter, P. J. and Samulski, R.J. (2000) J. Mol. Med. 6:17-27), rhinovirus, human immune deficiencyvirus (HIV), filovirus and engineered versions thereof (see, forexample, Cobinger G. P. et al (2001) Nat. Biotechnol. 19:225-30),virosomes, “naked” DNA liposomes, and nucleic acid coated particles, inparticular gold spheres. Particularly preferred are viral vectors likeadenoviral vectors or retroviral vectors (Lindemann et al. (1997) Mol.Med. 3:466-76 and Springer et al. (1998) Mol. Cell. 2:549-58). Liposomesare usually small unilamellar or multilamellar vesicles made ofcationic, neutral and/or anionic lipids, for example, by ultrasoundtreatment of liposomal suspensions. The DNA can, for example, beionically bound to the surface of the liposomes or internally enclosedin the liposome. Suitable lipid mixtures are known in the art andcomprise, for example, DOTMA(1,2-Dioleyloxpropyl-3-trimethylammoniumbromid) and DPOE(Dioleoylphosphatidyl-ethanolamin) which both have been used on avariety of cell lines.

Nucleic acid coated particles are another means for the introduction ofnucleic acids into cells using so called “gene guns”, which allow themechanical introduction of particles into the cells. Preferably theparticles itself are inert, and therefore, are in a preferred embodimentmade out of gold spheres.

In a further aspect the polynucleotide of the present invention isoperatively linked to expression control sequences allowing expressionin prokaryotic and/or eukaryotic host cells. Thetranscriptional/translational regulatory elements referred to aboveinclude but are not limited to inducible and non-inducible,constitutive, cell cycle regulated, metabolically regulated promoters,enhancers, operators, silencers, repressors and other elements that areknown to those skilled in the art and that drive or otherwise regulategene expression. Such regulatory elements include but are not limited toregulatory elements directing constitutive expression like, for example,promoters transcribed by RNA polymerase III like, e.g., promoters forthe snRNA U6 or scRNA 7SK gene, the cytomegalovirus hCMV immediate earlygene, the early or late promoters of SV40 adenovirus, viral promoter andactivator sequences derived from, e.g., NBV, HCV, HSV, HPV, EBV, HTLV,MMTV or HIV; which allow inducible expression like, for example, CUP-1promoter, the tet-repressor as employed, for example, in the tet-on ortet-off systems, the lac system, the trp system; regulatory elementsdirecting tissue specific expression, preferably nerve cell specificexpression, e.g. promoter (e.g. Thy-1.2, NSE, myosin light chain II,tyrosine hydroxylase, CaMKIIalpha promoter, platelet-derived growthfactor beta-chain (PDGF), dopamine beta-hydroxylase, Tau, regulatoryelements (e.g. NRSE/RE-1; neuron-restrictive silencing element/repressorelement 1) directing cell cycle specific expression like, for example,cdc2, cdc25C or cyclin A; or the TAC system, the TRC system, the majoroperator and promoter regions of phage A, the control regions of fd coatprotein, the promoter for 3-phosphoglycerate kinase, the promoters ofacid phosphatase, and the promoters of the yeast α- or a-mating factors.

As used herein, “operatively linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

Similarly, the polynucleotides of the present invention can form part ofa hybrid gene encoding additional polypeptide sequences, for example, asequence which encodes a protein that functions as a marker or reporter.The hybrid gene can lead to a fusion protein or the two or more partscan be separated by internal ribosomal entry sites (IRES) sequence,which lead to the expression of two or more separate proteins. Examplesof marker and reporter genes include β-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferace (neo¹, G418²), dihydrofolate reductase (DHFR),hygromysin-B-phosphotransferase (HPH), thymidine kinase (TK). lacZ(encoding β-galactosidase), green fluorescent protein (GFP) and variantsthereof and xanthine guanine phosphoribosyltransferace (XGPRT). As withmany of the standard procedures associated with the practice of theinvention, skilled artisans will be aware of additional useful reagents,for example, additional sequences that can serve the function of amarker or reporter. If the expression of the hybrid gene leads to onepolypeptide the hybrid polypeptide will usually include a first portionand a second portion; the first portion being a EPO variant polypeptideand the second portion being, for example, the reporter described aboveor an Ig constant region or part of an Ig constant region, e.g., the CH2and CH3 domains of IgG2a heavy chain. Other hybrids could include aheterologous peptide sequence to facilitate purification and/ordetection, e.g. an antigenic tag like, for example, a myc tag, or a tagwith preferential binding to a region, e.g. chitin taq or His tag.Recombinant nucleic acid molecules can also contain a polynucleotidesequence encoding a EPO variant polypeptide operatively linked to aheterologous signal sequence. Such signal sequences can direct theprotein to different compartments within the cell and are well known tosomeone of skill in the art. A preferred signal sequence is a sequencethat facilitates secretion of the resulting protein. Preferably thesesignal and/or taq sequences are designed in such that they can becleaved of the EPO variant after purification to provide an essentiallypure protein without two many amino acids, preferably not more than 10additional amino acids to the final EPO. Such cleavage sites are wellknown in the art and comprise, e.g endopeptidase cleavage sites andintein cleavage sites.

Another aspect of the present invention is a host cell geneticallyengineered with the polynucleotide or the vector as outlined above. Thehost cells that may be used for purposes of the invention include butare not limited to prokaryotic cells such as bacteria (for example. Ecoli and B. subtilis), which can be transformed with, for example,recombinant bacteriophage DNA, placid DNA, or cosmid DNA expressionvectors containing the polynucleotide molecules of the invention; simpleeukaryotic cells like yeast (for example, Saccharomyces and Pichia),which can be transformed with, for example, recombinant yeast expressionvectors containing the polynucleotide molecule of the invention; insectcell systems like, for example, Sf9 of Hi5 cells, which can be infectedwith, for example, recombinant virus expression vectors (for example,baculovirus) containing the polynucleotide molecules of the invention;Xenopus oocytes, which can be injected with, for example, plasmids;plant cell systems, which can be infected with, for example, recombinantvirus expression vectors (for example, cauliflower mosaic vires (CaMV)or tobacco mosaic virus (TMV)) or transformed with recombinant plasmidexpression vectors (for example, Ti plasmid) containing a EPO variantnucleotide sequence; or mammalian cell systems (for example, COS, CHO,BHK, HEK293, VERO, HeLa, MDCK, Wi38, Swiss 3T3 and NIH 3T3 cells), whichcan be transformed with recombinant expression constructs containing,for example, promoters derived, for example, from the genome ofmammalian cells (for example, the matallothionein promoter) frommammalian viruses (for example, the adenovirus late promoter, CMV IE andthe vaccinia virus 7.5K promoter) or from bacterial cells (for example,the tet-repressor binding is employed in the tet-on and tet-offsystems). Also useful as host cells are primary or secondary cellsobtained directly from a mammal sod transfected with a plasmid vector orinfected with a viral vector. Depending on the host cell and therespective vector used to introduce the polynucleotide of the inventionthe polynucleotide can integrate, for example, into the chromosome orthe mitochondrial DNA or can be maintained extrachromosomally like, forexample, episomally or can be only transiently comprised in the cells.

Since EPO is heavily glycosylated in vivo it is desirable to chose anexpression system, which provides faithful glycosylation of the protein.Consequently, it is preferred to introduce the polynucleotides encodingthe EPO slice variants of the present invention into higher eukaryoticcells, in particular into mammalian cells, e.g. COS, CHO, BHK, HEK293,VERO, HeLa, MDCK, Wi38, Swiss 3T3 or NIH 3T3 cells.

A further aspect of the present invention is a transgenic non-humananimal containing a polynucleotide, a vector and/or a host cell asdescribed above. The animal can be a mosaic animal, which means thatonly part of the cells making up the body comprise polynucleotides,vectors, and/or cells of the present invention or the animal can be atransgenic animal which means that all cells of the animal comprise thepolynucleotides and/or vectors of the present invention or are derivedfrom a cell of the present invention. Mosaic or transgenic animals canbe either homo- or heterozygous with respect to the polynucleotides ofthe present invention contained in the cell. In a preferred embodimentthe transgenic animals are either homo- or heterozygous knock-out orknock-in animals with respect to the genes which code for the proteinsof the present invention. The animals can in principal be any animal,preferably, however, it is a mammal, selected from the group ofnon-human primate horse, bovine, sheep, goat, pig, dog, cat, goat,rabbit, mouse, rat, guinea pig, hamster, or gerbil.

Another aspect of the present invention is a process for producing anEPO variant polypeptide encoded by a polynucleotide of the presentinvention comprising: culturing the host cell described above andrecovering the polypeptide encoded by said polynucleotide. Preferredcombinations of host cells and vectors are outlined above the furthercombination will be readily apparent to someone of skill in the art.Depending on the intended later use of the recovered peptide a suitablecell type can be chosen. As outlined above eukaryotic cells arepreferably chosen, if it is desired that the proteins produced by thecells exhibit an essentially natural pattern of glycosylation andprokaryotic cells are chosen, if, for example, glycosylation or othermodifications, which are normally introduced into proteins only ineukaryotic cells, are not desired or not needed.

It is known in the prior art that the pharmacokinetic of protein drugscan be significantly altered by modification of the protein. For fulllength EPO it has been described that glycosylation, in particular thepresence of sialic acid residues at the end of the oligosaccharide sidechains attributes to the circulation time (WO 95/05465) and that removalof sialic acid groups exposes galactose residues, which increasesclearance by the liver. Therefore, one approach taken to enhance EPOcirculation time was the increase in sialic acid residues. Severalapproaches, thus, involve the provision of additional glycosylationsites (see e.g. WO 91/05867, WO 94/09257 and WO 01/81405. Such modifiedEPO analogs may have at least one additional N-linked and/or O-linkedcarbohydrate chain. Other attempts to improve the half life of EPOinvolved the addition of polyethylene glycol residues (PEG) of varyinglength the amino acid backbone (see e.g. WO 00/32772, WO 01/02017, WO03/029291. Another attempt used the modification of EPO molecules withat least one N-linked and/or O-linked oligosaccharide which were furthermodified with oxidation, sulfation, phosphorylation PEGylation or acombination thereof (see WO 2005/025606. All these approaches canequally be employed to extend the half life of the EPO variants of thepresent invention and accordingly in a preferred embodiment aboveprocess further comprising the step of modifying the EPO variant,wherein the modification is selected from the group consisting ofoxidation, sulfation, phosphorylation, addition of oligosaccharides orcombinations thereof. If the addition of further N-linked or O-linkedoligonucleotides is desired it is possible to introduce them byintroducing additional glycosylation sites as has been described in theprior art, e.g. at positions 30, 51, 57, 69, 88, 89, 136 and/or 138, ifthe respective position is present in the variant of the presentinvention (see WO 01/81405).

A further aspect of the invention is a process for producing cellscapable of expressing at least one of the EPO variants comprisinggenetically engineering cells in vitro with the vector of claim 3 or 4,wherein, said EPO variant polypeptide(s) is(are) encoded by apolynucleotide of the present invention.

Another aspect of the invention is a polypeptide having the amino acidsequence encoded by a polynucleotide of the invention or obtainable bythe process mentioned above. The polypeptides of the invention includeall those disclosed herein and fragments of these polypeptides, whichcarry between 1 and 10 N- and/or C-terminal deletions. Preferably, thedeletions are less than 10, less than 9, less than 8, less than 7, lessthan 8, less than 7, less than 6, less than 5, less than 4, less than 3,less than 2, less than 1 amino acids. The polypeptides embraced by theinvention also include fusion proteins that contain either the EPO slicevariant as indicated in SEQ ID Nos 2, 4, 6, 8, 10, 12, 14, 16, 18, 20and 22 or humanized version of 14, 16, 18, 20 and 22 or a fragmentthereof as defined above fused to an unrelated amino acid sequence. Theunrelated sequences can comprise additional functional domains or signalpeptides. Signal peptide are described in greater detail and exemplifiedbelow.

The polypeptides can be any of those described above but with not morethan 10 (e.g., not more than: 10, nine, eight, seven, six, five, four,three, two, or one) conservative substitutions. Conservativesubstitutions are known in the art and typically include substitutionof, e.g. one polar amino acid with another polar amino acid and oneacidic amino acid with another acidic amino acid. Accordingly,conservative substitutions preferably include substitutions within thefollowing groups of amino acids: glycine, alanine valine, proline,isoleucine, and leucine (non polar, aliphatic side chain); aspartic acidand glutamic acid (negatively charged side chain); asparagine,glutamine, methionine, cysteine, serine and threonine (polar unchargedside chain); lysine, histidine and arginine; and phenylalanine,tryptophane and tyrosine (aromatic side chain); and lysine, arginine anhistidine (positively charged side chain). It is well known in the arthow to determine the effect of a given substitution, e.g. on pK₁ etc.All that is required of a polypeptide having one or more conservativesubstitutions is that it has at least 50% (e.g. at least: 55%; 60%; 65%,70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 99.5%; or 100% or more) of theability of the unaltered EPO variant to protect neurons from damage/celldeath (e.g. by apoptosis or necrosis), wherein the cell death is inducedby oxygen and/or glucose deprivation, by toxic, chemical, physical,mechanical, inflammatory or radiation exposure or by viral or bacterialinfection.

Both polypeptides and peptides can be produced by standard in vitrorecombinant DNA techniques and in vivo transgenesis, using nucleotidesequences encoding the appropriate polypeptides or peptides. Methodswell-known to those skilled in the art can be used to constructexpression vectors containing relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example, thetechniques described in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y.; 1989], andAusubel et al., Current Protocols in Molecular Biology [Green PublishingAssociates and Wiley Interscience, N.Y., 1989].

Polypeptides and fragments of the invention also include those describedabove, but modified for in vivo use by the addition, at the amino-and/or carboxyl-terminal ends, of blocking agents to facilitate survivalof the relevant polypeptide in vivo. This can be useful in thosesituations in which the peptide termini tend to be degraded by proteasesprior to cellular uptake. Such blocking agents can include, withoutlimitation, additional related or unrelated peptide sequences that canbe attached to the amino and/or carboxyl terminal residues of thepeptide to be administered. This can be done either chemically duringthe synthesis of the peptide or by recombinant DNA technology by methodsfamiliar to artisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

The term “isolated” polypeptide or peptide fragment as used hereinrefers to a polypeptide or a peptide fragment which either has nonaturally-occurring counterpart or has been separated or purified fromcomponents which naturally accompany it, e.g., in tissues such astongue, pancreas, liver, spleen, ovary, testis, muscle, joint tissue,neural tissue, gastrointestinal tissue or tumor tissue, or body fluidssuch as blood, serum, or urine. Typically, the polypeptide or peptidefragment is considered “isolated” when it is at least 70%, by dryweight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated. Preferably, apreparation of a polypeptide (or peptide fragment thereof) of theinvention is at least 80% more preferably at least 90%, and mostpreferably at least 99%, by dry weight, the polypeptide (or the peptidefragment thereof), respectively, of the invention. Thus, for example, apreparation of polypeptide x is at least 80%, more preferably at least90%, and most preferably at least 99%, by dry weight, polypeptide x.Since a polypeptide that is chemically synthesized is, by its nature,separated from the components that naturally accompany it, the syntheticpolypeptide is “isolated.”

An isolated polypeptide (or peptide fragment) of the invention can beobtained, for example, by extraction from a natural source (e.g., fromtissues or bodily fluids); by expression of a recombinant nucleic acidencoding the polypeptide; or by chemical synthesis. A polypeptide thatis produced in a cellular system different from the source from which itnaturally originates is “isolated,” because it will necessarily be freeof components which naturally accompany it. The degree of isolation orpurity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A further aspect of the invention is an antibody, which specificallybinds to the EPO variant polypeptide encoded by polynucleotides of theinvention or obtainable by the process mentioned above. The term“antibody” comprises monoclonal and polyclonal antibodies and bindingfragments thereof, in particular Fc-fragments as well as so called“single-chain-antibodies” (Bird R. E. et al. (1988) Science 242:423-6),chimeric, humanized, in particular CDR-grafted antibodies, and dia ortetrabodies (Holliger P. et al (1993) Proc. Natl. Acad. Sci. U.S.A.90:6444-8). Also comprised are immunoglobulin like proteins that areselected through techniques including, for example, phage display tospecifically bind to the polypeptides of the present invention. In thiscontext the term “specific binding” refers to antibodies raised againstpeptides derived from splice junctions or junctions created by otherprocesses, e.g. ENIT|RVGQ of hS3, VGQQ|ALLV of hs1-4, VNFY|ALLY of h1-5,KRME|PWEP of hS4, ITVP|GPVG of h1-1, LNEN|NHC of h2-1, KRME|KELM of mS,LLAN|FLRG of mG3, DTFC|RRGD of mG5, KVNF|LRGK of m301 or LSEA|VHGR ofmK3. Such peptides can comprise additional or less N- or C-terminalamino acids. An antibody is considered to be specific to the EPOvariant, if its affinity towards the variant it at least 50-fold higher,preferably 100-fold higher, more preferably at least 1000-fold higherthan towards the full length human or murine EPO. Preferably specificantibodies of the present invention do not or essentially do not bind tofull length human or marine EPO. It is well known in the art how to makeantibodies and to select antibodies with a given specificity.

A further aspect of the present invention concerns the use of apolynucleotide, a vector, a host cell or a polypeptide of the presentinvention for the manufacture of a medicament for the treatment orprevention of a condition associated with tissue damage due to celldeath (e.g. apoptosis and necrosis). The apoptosis or necrosis leads tothe cell destruction, which can be prevented or ameliorated when usingthe polynucleotide, vector, host cell or polypeptide of the presentinvention. Cell death can be induced by many different internal andexternal stimuli and include preferably ischemia, hypoxia, bacterial orviral infection, radiation, or induced by metabolic, toxic, chemical,autoimmunologic, or traumatic stimuli. It is well known in the art howto detect cell death like, for example, using morphological criteria, aTUNNEL assay, MTT-assay, life/death assay by staining (e.g. Ethidiumbromide and acridine orange staining), caspase assay, electronmicroscopy, DNA-laddering or the LDH release assay described below. Forexample, apoptosis is characterized by chromatin fragmentation,extravasation of cellular contents and eventually death of the cell. Ithas been recognized to play a role in many acute or chronic pathologicprocesses. Accordingly, a preferred use of the present inventioncomprises the administration of polynucleotides, vectors, host cells orpolypeptides of the present invention to prevent, treat or ameliorateacute and chronic neurodegenerative or neuroinflammatory disorders,acute or chronic disorder of the heart (e.g. myocardial infarction),lung (e.g. asthma, chronic obstructive lung disease), kidney (e.g.glomerulonephritis), liver (e.g. chronic liver failure) or pancreas(e.g. pancreatitis), as well as conditions associated with cell (e.g.stem cell) or organ transplantation (e.g. kidney or liver). In thisrespect is also envisioned that the EPO variants of the presentinvention can be included in storage solutions used for storing organsor limbs for transport and/or after traumatic injury.

Acute neurodegenerative disorders include, but are not limited to,various types of acute neurodegenerative disorders associated withneuronal cell death including cerebrovascular insufficiency, focal ordiffuse brain trauma, diffuse brain damage, and spinal cord injury.Examples of acute neurodegenerative disorders are: cerebral ischemia orinfarction including embolic occlusion and thrombotic occlusion,reperfusion following acute ischemia, perinatal hypoxic-ischemic injury,cardiac arrest, as well as intracranial hemorrhage of any type (such asepidural, subdural, subarachnoid and intracerebral), and intracranialand intravertebral lesions (such as contusion, penetration, shear,compression and laceration), whiplash shaken infant syndrome infectiousencephalitis (e.g. herpes encephalitis), meningitis (e.g. bacterial),headache (e.g. migraine).

Chronic neurodegenerative disorders that can be treated with the EPOvariants of the present invention include, but are not limited to,dementias (e.g. Alzheimer's disease, vascular dementias), Pick'sdisease, diffuse Lewy body disease, progressive supranuclear palsy(Steel-Richardson syndrome), multiple sclerosis, multiple system atrophy(including Shy-Drager syndrome), chronic epileptic conditions associatedwith neurodegeneration, motor neuron diseases including amyotrophiclateral sclerosis, degenerative ataxias, cortical basal degeneration,ALS-Parkinson's-Dementia complex of Guam, subacute sclerosingpanencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies (including multiple system atrophy), primaryprogressive aphasia, striatonigral degeneration, Machado-Josephdisease/spinocerebellar ataxia type 3 and olivopontocerebellardegenerations, Gilles De La Tourette's disease, bulbar and pseudobulbarpalsy, spinal and spinobulbar muscular atrophy (Kennedy's disease),primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmanndisease, Kugelberg-Welander disease, Tay-Sach's disease, Sandhoffdisease, familial spastic disease, spastic paraparesis, progressivemultifocal leukoencephalopathy, familial dysautonmia (Riley-Daysyndrome), and prion diseases (including, but not limited toCreutzfeldt-Jakob, Gerstmann-Strussler-Scheinker disease, Kuru fatalfamilial insomnia), polyneuropathies (e.g. diabetic, alcohol-toxic,Guillain-Barré-Syndrome, chronic inflammatory demyelinatingpolyneuropathy), prion diseases, addiction, affective disorders (e.g.depression), schizophrenic disorders, chronic fatique syndrome, chronicpain (e.g. lower back pain).

A further aspect of the present invention concerns the use of apolynucleotide, a vector, a host cell or a polypeptide of the presentinvention for the manufacture of an anti-aging medication. The basis forthis application of the EPO variants of the present invention is thefact that the progressing deterioration of most bodily functions, whichaccompanies aging has been associated with cell death and it is,therefore, envisioned that the EPO variants of the present invention,which only provide the beneficial cell protective effect can be takencontinuously without suffering the side effects usually associated withthe contnious administration of EPO, which, however, can be attributedto the erythropoietic effect of EPO.

The inventors have astonishingly found that the nucleic acids andproteins according to the invention possess astonishinganti-inflammatory properties (see figures and experiments). Thus, theseEPO variants are useful in treatment of inflammatory and degenerativediseases. Inflammatory diseases are deceases such as but not limited tomultiple sclerosis, viral and bacterial infections or sepsis.Degenerative diseases are diseases such as but not limited to stroke,myocardial infarctions.

The invention also relates to all kinds of forms of in vivo expressionof the nucleic acids according to the invention. It further relates totransformed cells, in particular stem cells which are used astherapeutic agents. Such cells may be stably transformed with a nucleicacid according to the invention. The nucleic acid may in a cassettewhere it is operably linked to a promoter. The promoter may capable ofdriving the expression only in particular tissues, such as but notlimited to neuronal tissue or the brain or tissue which exhibitsinflammation or degeneration. Respective teaching may be taken fro, WO97/14307.

the activity (in units) of EPO polypeptide is traditionally definedbased on its effectiveness in stimulating red cell production in rodentmodels (and as derived by international standards of EPO). One unit (U)of regular EPO (MW of about 34,000) is about 10 ng of proton) (1 mgprotein is approximately 1000,000 U). However, as mentioned theinvention involves the use of non-hematopoietic forms of erythropoietin,and as such, this definition based on hematopoietic activity isinappropriate. Thus, as used herein, the activity unit of EPO variant isdefined as the amount of protein required to elicit the same activity inneural or other erythropoietin-responsive cellular systems as iselicited by native EPO in the same system. The skilled artisan willreadily determine the units of a non-hematopoietic EPO following theguidance herein.

In a further aspect the present invention provides a pharmaceuticalcomposition comprising a polynucleotide, a vector, a host cell, apolypeptide and/or an antibody of the present invention and a one ormore pharmaceutically acceptable carrier.

In the practice of one aspect of the present invention, a pharmaceuticalcomposition as described above may be administered to a mammal by anyroute which provides a sufficient level of an erythropoietin variant. Itcan be administered systemically or locally. Such administration may beparenterally, transmucosally, e.g., orally, nasally, rectally,intravaginally, sublingually, submucosally or transdermally. Preferably,administration is parenteral, e.g. via intravenous or intraperitonealinjection, and also including, but is not limited to, intra-arterial,intramuscular, intradermal and subcutaneous administration. If thepharmaceutical composition of the present invention is administeredlocally it can be injected directly into the organ or tissue to betreated. In cases of treating the nervous system this administrationroute includes, but is not limited to, the intracerebral,intraventricular, intracerebroventricular, intrathecal, intracistemal,intraspinal and/or peri-spinal routes of administration, which canemploy intracranial and intravertebral needles, and catheters with orwithout pump devices.

In a preferred embodiment of pharmaceutical composition comprises an EPOvariant polypeptide in a dosage unit form adapted for protection orenhancement of EPO-responsive cells, tissues or organs which comprises,per dosage unit, an effective non-toxic amount within the range fromabout 0.5 mg to 5 mg of EPO variants; 0.6 mg to 5 mg of EPO variants;0.7 mg to 5 mg of EPO variants; 0.8 mg to 5 mg of EPO variants; 0.9 mgto 5 mg of EPO variants; 1 to 5 mg of EPO variants; 1.5 to 5 mg of EPOvariants; 2 to 5 mg of EPO variants; 2.5 to 5 mg of EPO variants; 3.5 to5 mg of EPO variants; 4 mg to 5 mg of EPO variants; or 4.5 to 5 mg ofEPO variants and a pharmaceutically acceptable carrier.

In a preferred embodiment, an EPO variant polypeptide may beadministered systemically at a dosage between 100 nanograms to about 50micrograms per kg body weight, preferably about 20 micrograms to about30 micrograms per kg-body weight. Such serum levels may be achieved atabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours post-administration. Suchdosages may be repeated as necessary. For example, administration may berepeated daily, or every other, third, fourth, fifth, sixth, or seventhday, as long as clinically necessary, or after an appropriate interval,e.g., every 1 to 12 weeks, preferably, every 3 to 8 weeks. In oneembodiment, the effective amount of EPO variant and a pharmaceuticallyacceptable carrier may be packaged in a single dose vial or othercontainer. Depending on the respectively treated disease or conditionthe EPO variant can be administered in a single dose, for apredetermined period of time or continuously. When an acute condition ordisease is treated it might be sufficient to provide the patient with asingle dose of EPO variant or for a period of, e.g. for 2 days to 12months, preferably 1 week to 6 months, more preferably 2 weeks to 3months. If a chronic disease or condition is treated or if the EPOvariant is used to prevent or reduce the deterioration associated withaging the EPO variant can be administered continuously. If the EPOvariant of the present invention is administered for a given time periodor continuously it is preferably administered in the intervals andpreferred intervals indicated above. The intervals necessary will dependin part on the serum level of the EPO variant necessary to treat orameliorate the respective disease and on the pharmacokinetic of therespective EPO variant, which will in part depend on modifications ofEPO by, for example, PEG. It will be in the discretion of thepractitioner to determine the exact duration, dose and type of EPOvariant taking into consideration, e.g. the condition of the patient tobe treated, the severity of the dominion etc.

For other routes of administration, such as by use of a perfusate,injection into an organ, or other local administration, a pharmaceuticalcomposition will be provided which results in similar levels of an EPOvariant as described above. A level of about 10 pg/ml to about 1000ng/ml is desired.

The pharmaceutical compositions of the invention may comprise atherapeutically effective amount of a compound, e.g. polynucleotide,polypeptide, cell or vector, and a pharmaceutically acceptable carrier.In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as saline solution in water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, such aspeanut oil, soybean oil, mineral oil, sesame oil and the like. A salinesolution is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. The compounds ofthe invention can be formulated as neutral or salt forms.Pharmaceutically acceptable salt include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, trethylamine, 2-ethylamino ethanol,histidine, procaine, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified from, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for oral administration may beprovided as capsules or tablets; as powders or granules; as solutions,syrups or suspensions (in aqueous or non-aqueous liquids); as ediblefoams or whips; or as emulsions. Tablets or hard gelatine capsules maycomprise lactose, starch or derivatives thereof, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, stearic acid or saltsthereof. Soft gelatine capsules may comprise vegetable oils, waxes,fats, semi-solid, or liquid polyols etc. Solutions and syrups maycomprise water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract (e.g., glycerylmonostearate or glyceryl distearate may be used). Thus, the sustainedrelease of an active agent may be achieved over many hours and, ifnecessary, the active agent can be protected from being degraded withinthe stomach. Pharmaceutical compositions for oral administration may beformulated to facilitate release of an active agent at a particulargastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.Pharmaceutical compositions adapted for topical administration may beprovided as ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. For topical administration tothe skin, mouth, eye or other external tissues a topical ointment orcream is preferably used. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops. In these compositions, the active ingredient can be dissolved orsuspended in a suitable carrier, e.g., in an aqueous solvent.Pharmaceutical compositions adapted for topical administration in themouth include lozengers, pastilles and mouthwashes.

Pharmaceutical compositions adapted for nasal administration maycomprise solid carriers such as powders (preferably having a particlesize in the range of 20 to 500 microns). Powders can be administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nose from a container of powder held close to the nose.Alternatively, compositions adopted for nasal administration maycomprise liquid carriers, e.g., nasal sprays or nasal drops. Thesecompositions may comprise aqueous or oil solutions of the activeingredient. Compositions for administration by inhalation may besupplied in specially adapted devices including, but not limited to,pressurized aerosols, nebulizers or insufflators, which can beconstructed so as to provide predetermined dosages of the activeingredient. In a preferred embodiment, pharmaceutical compositions ofthe invention are administered via the nasal cavity to the lungs.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which nay contain antioxidants, buffers, bacteriostats andsolutes that render the compositions substantially isotonic with theblood of an intended recipient. Other components that may be present insuch compositions include water, alcohols, polyols, glycerine andvegetable oils, for example. Compositions adapted for parenteraladministration may be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, e.g., sterile saline solution for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.In one embodiment, an autoinjector comprising an injectable solution ofan EPO variant may be provided for emergency use by ambulances,emergency rooms, and battlefield situations, and even forself-administration in a domestic setting, particularly where thepossibility of traumatic amputation may occur, such as by imprudent useof a lawn mower. The likelihood that cells and tissues in a severed footor toe will survive after reattachment may be increased by administeringan EPO variant to multiple sites in the severed part as soon aspracticable, even before the arrival of medical personnel on site, orarrival of the afflicted individual with severed toe in tow at theemergency room.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically-sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile saline can be providedso that the ingredients may be mixed prior to administration.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

A perfusate composition may be provided for use in transplanted organbaths, for in situ perfusion, or for administration to the vasculatureof an organ donor prior to organ harvesting.

Such pharmaceutical compositions may comprises levels of an EPO variantor a form of an EPO variant not suitable for acute or chronic, local orsystem administration to an individual. but will serve the functionsintended herein in a cadaver, organ bath, organ perfusate, or in situperfusate prior to removing or reducing the levels of the EPO variantcontained therein before exposing or returning the treated organ ortissue to regular circulation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In another embodiment, for example, EPO variant can be delivered in acontrolled-release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Sefton (1997) CRC Crit. Ref. Biomed.Eng. 14: 201; Buchwald et al. (1980) Surgery 88:507; Saudek et. al.(1989) N. Eng. J. Med. 321: 574). In another embodiment, the compoundcan be delivered in a vesicle, in particular a liposome (see Langer(1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, N.Y., 353-365; WO 91/04014; U.S. Pat. No. 4,704,355). Inanother embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release (1974) Langer and Wise (eds.), CRCPress: Boca Raton, Fla.; Controlled Drug Bioavailability, Drug ProductDesign and Performance, (1984) Smolen and Ball (eds.), Wiley: N.Y.;Ranger and Peppas (1953) J. Macromol. Sci. Rev. Macromol. Chem. 23: 61;see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann.Neurol. 25: 351; Howard et al. (1989) J. Neurosurg. 71: 105).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the target cells, tissue ororgan, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson (1984) 115-138 in Medical Applications of Controlled Release,vol. 2). Other controlled release systems are discussed in the review byLanger (1990, Science 249: 1527-1533)

In another embodiment, EPO variant, as properly formulated, can beadministered by nasal, oral, rectal, vaginal, or sublingualadministration.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, far example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as silastic membranes, or fibers.

Selection of the preferred effective dose will be determined by askilled artisan based open considering several factors which will beknown to one of ordinary skill in the art. Such factors include theparticular form of the pharmaceutic composition, e.g. polypeptide orvector, and its pharmacokinetic parameters such as bioavailability,metabolism, half-life, etc., which will have been established during theusual development procedures typically employed in obtaining regulatoryapproval for a pharmaceutical compound. Further factors in consideringthe dose include the condition or disease to be treated or the benefitto be achieved in a normal individual, the body mass of the patient, theroute of administration, whether administration is acute or chronic,concomitant medications, and other factors well known to affect theefficacy of administered pharmaceutical agents. Thus the precise dosageshould be decided according to the judgment of the practitioner and eachpatient's circumstances, e.g., depending upon the condition and theimmune status of the individual patient, according to standard clinicaltechniques.

In another aspect of the invention, a perfusate or perfusion solution isprovided for perfusion and storage of organs for transplant, theperfusion solution including an amount of an pharmaceutic compositionseffective to protect EPO variant-responsive cells and associated cells,tissues or organs.

Transplant includes but is not limited to xenotransplantation, where aorgan (including cells, tissue or other bodily part) is harvested fromone donor and transplanted into a different recipient; andautotransplant, where the organ is taken from one part of a body andreplaced at another, including bench surgical procedures, in winch anorgan may be removed, and while ex vivo, resected, repaired, orotherwise manipulated, such as for tumor removal, and then returned tothe original location. In one embodiment, the perfusion solution is theUniversity of Wisconsin (UW) solution (U.S. Pat. No. 4,798,824) whichcontains from about 1 to about 25 U/ml erythropoietin, 5% hydroxyethylstarch (having a molecular weight of from about 200,000 to about 300,000and substantially free of ethylene glycol, ethylene chlorohydrin, sodiumchloride and acetone); 25 mM KH₂PO₄; 3 mM glutathione; 5 mM adenosine;10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5 mMCaCl₂. 105 mM sodium gluconate; 200,000 units penicillin; 40 unitsinsulin; 16 mg Dexamethasone; 12 mg Phenol Red; and has a pH of 7.4-7.5and an osmolality of about 320 mOSm/l. The solution is used to maintaincadaveric kidneys and pancreases prior to transplant. Using thesolution, preservation may be extended beyond the 30-hour limitrecommended for cadaveric kidney preservation. This particular perfusateis merely illustrative of a number of such solutions that may be adaptedfor the present use by inclusion of an effective amount of thepharmaceutical composition. In a further embodiment, the perfusatesolution contains the equivalent from about 5 to about 35 U/mlerythropoietin, or from about 10 to about 30 U/ml erythropoietin.

While the preferred recipient of an EPO variant for the purposes hereinthroughout is a human, the methods herein apply equally to othermammals, particularly domesticated animals, livestock, companion and zooanimals. However, the invention is not so limiting and the benefits maybe applied to any mammal.

If a person is known to be at risk of developing a stroke a prophylacticadministration of the pharmaceutical composition of the presentinvention is possible. In these cases the pharmaceutical compositions,in particular EPO variant polypeptide is preferably administered inabove outlined preferred and particular preferred doses on a dailybasis. Preferably, between 100 nanograms to about 50 micrograms per kgbody weight, preferably about 20 micrograms to about 50 micrograms perkg-body weight. This administration can be continued until the risk ofdeveloping a stroke has lessened. In most instances, however, thepharmaceutical composition will be administered once a stroke has beendiagnosed. In these cases it is preferred that a first dose of thepharmaceutical composition is administered for the first time within 24hours after the first symptoms of a stroke are evident, preferablywithin 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour or less. Preferablythe administration is then continued for preferably at least 7, morepreferably at least 14 and more preferably for at least 21 days. Thedoses are administered preferably once a day and preferably in aboveindicated doses.

BRIEF DESCRIPTION OF THE FIGURES AND DRAWINGS

FIG. 1A: the DNA products of various PCR reactions performed with eitherpure plasmid comprising the different murine EPO variants or cDNA frommouse brain of kidney, which are separated on a 1.2% agarose gel. Fromthe left to the right the lanes comprise: 1 kb molecular weight marker,the product of pure mK3, pure mG3, pun mGS, pure m301, pure mS, puremWT, brain cDNA, kidney cDNA. FIG. 1B: the DNA product of a PCRperformed with cDNA from human brain. From the left to the right thelanes comprise 1 kb molecular weight standard and the PCR product ofhuman braincDNA.

FIG. 2A: Alignment of nucleotide sequences of the EPO variantsidentified in murine brain cDNA and “wild type” murine EPO, i.e. thesequence of the previously described EPO; FIG. 2B: Alignment ofnucleotide sequences of the EPO variants identified in murine brain cDNAand “wild type” murine EPO, i.e. the sequence of the previouslydescribed EPO (continued from FIG. 2A).

FIG. 3A-1: Alignment of nucleotide sequences of the EPO variantsidentified in human braincDNA and “wild type” human EPO; FIG. 3A-2:Alignment of nucleotide sequences of the EPO variants identified inhuman brain cDNA and “wild type” human EPO (continued from FIG. 3A-1);FIG. 3B: Alignment of nucleotide sequences of the EPO variantsidentified in human brain cDNA and “wild type” human EPO (continued fromFIG. 3A-1).

FIG. 4: Alignment of the amino acid sequences of the BPO variantsidentified in mouse and human with the respective “wild type” EPO.

FIG. 5A: Hematopoietic activity of murine and human EPO and the EPOvariants of the present invention shown as results of a colony formingassay for murine EPO and EPO variants. FIG. 5B: Hematopoietic activityof murine and human EPO and the EPO variants of the present inventionshown as results of a colony forming assay for human EPO and EPOvariants.

FIG. 6: Experimental setup for neuroprotection assays with rhEPO andEPO-isomers.

FIG. 7A: an experiment with 1 h 40 min and I h 50 min oxygen glucosedeprivation (OGD) length. At both time-points a protection rate of40-50% for the neuroguardians, but no protection with mEPO and rhEPO wasobserved. FIG. 7B: an experiment with two different time-points (OGDlength between two experiments varies according to density of neurons).At 2 h 45 min only weak protection is achieved with wtEPO (20-30%)compared to neuroguardians (60-70%). Full protection capacity of rb EPOis only observed at higher damage levels (3. h 15 min).

FIG. 8A: an experiment with 2 h 00 mi 2 h 15 min and 2 h 20 min OGDlength with a protein concentration equalling 100 U/l hEPO. At all threetime-points a protection rate of 40-50% for the-human, but no protectionwith mEPO and rhEPO. FIG. 8B: an experiment with two differenttime-points (OGD length between two ex-periments varies according todensity of neurons). At 2 h 45 min only weak protection is achieved withwtEPO (20-30%) compared to neuroguardians (60-70%). Full protec-tioncapacity of EPO is only observed at higher damage levels (3 h 15 min).

FIG. 9A: a Western Blot from medium of HEK293-cells transfected withpcDNA3.1-V5/His-hEPO, pcDNA3.1-V5/His-hS3 or pcDNA3.1-V5/His-hS4,respectively. These media were used for experiments shown in FIGS. 8Aand B. Concentration of hEPO, quantified by the mouse-EPO-ELISA(R&D) was2 U/mL. rhEPO (=2.5 ng was loaded on the gel), hEPO (=0.4 ng was loadedon the gel), hS3 and hS4: each 20 μL medium (collected 2 days aftertransfection). Marker=5 μL BenchMark™ His-tagged Protein Standard(Invitrogen). FIG. 9B: a Western Blot of His-Tag-purified mouse wildtype EPO (mEPO), human hS3 and hS4 EPO variants. mEPO was quantifiedwith the EPO-mouse-ELISA. 130 pg mEPO were loaded onto the gel. (primaryantibody: rabbit anti-rhEPO-Antikorper; Santa-Cruz).

FIG. 10A: Alignment of the amino acid sequences of the EPO variantscreated recombinantly (alpha-helix mutants) and identified in vivo.Herein SEQ ID NO: 50 is the human alpha helix wild type sequence; SEQ IDNO: 51 is hAmA (point mutation Alanin); SEQ ID NO: 52 is hAmE (pointmutation glutamic acid); SEQ ID NO: 53 is hA-10 (deletion mutant)and SEQID NO: 54 is hA-20 (deletion mutant); FIG. 10B: Alignment of the aminoacid sequences of the EPO variants created recombinantly (alpha-helixmutants) and identified in vivo. Herein SEQ ID NO: 50 is the human alphahelix wild type sequence; SEQ ID NO: 51 is hAmA (point mutation Alanin);SEQ ID NO: 52 is hAmE (point mutation glutamic acid); SEQ ID NO: 53 ishA-10 (deletion mutant) and SEQ ID NO: 54 is hA-20 (deletion mutant)(continued from FIG. 10A).

FIG. 11A: Column diagram representing the average values of normalizedLDH release resulting from hEPO and hS3 mediated cytoprotection in amodel of ischemia consisting of serum deprivation and hypoxia in H9c2cardiac myoblasts. H9c2 cells were incubated in serum-deprivedDMEM-medium either in normoxic or hypoxic conditions for 24 h. Apoptosiswas assessed 24 h later by LDH assay. Data were normalized by settingthe delta LDH release of untreated cells in normoxic and hypoxicconditions to 100%. FIG. 11B: A box plot diagram showing the median(line across the box), the 25^(th) percentile (lower hinge), the 75thpercentile (upper hinge), the maximum and the minimum value ofnormalized LDH release resulting from hEPO and hS3 mediatedcytoprotection in a model of ischemia consisting of serum deprivationand hypoxia in H9c2 cardiac myoblasts. Number of experiments n=7.P*<0.001 (ANOVA1).

FIG. 12A: Immunoprecipitation of EPO variants using an anti-mEPOantibody from R&D (goat, biotin-labelled)-showing detection of a secondEPO isoform (30 .kDa) in a kidney protein extract of CoCl2-treated mice(129S6). FIG. 12B: Blocking of the antibody-antigen interaction betweenEPO variants and the anti-mEPO antibody by DarbpoietinA.

FIG. 13: Neuroprotection mediated by Erythropoietin Alpha-helix. (hA;n=4). hA I 00/UL: 30 pM; hA 50 U/L: 15 pM; hEPO: 30 pM=100 U/L; P*<0.05;ANOVA1 versus control.

FIG. 14: Neuroprotection mediated by several human EPO-isoforms (n=6)P*<0.05; ANOVA1 versus control

FIG. 15A: column diagram showing the average values of normalized LDHrelease mediated by Erythropoietin Alpha-helix deletion variants (n=6)P*<0.05; ANOVA1 versus control. FIG. 15B: Box plot showing the mediansand percentiles (25%, 75%) values of normalized LDH release medicated byErythropoietin Alpha-helix deletion variants.

FIG. 16. Effects of human EPO variants and full length human EPO onLPS-induced cytokine production by human macrophages. Purified humanmonocytes were differentiated into macrophages in the presence of rhuM-CSF (50 ng/mL) for 6 days. Macrophages (I×106/mL) were pre-incubatedwith hS4, hS3 or hEPO (300 mU/mL each) for 3 h and then stimulated with10 ng/mL endotoxin (LPS from E.coli 0127:B8) for 4 h. Cytokineconcentration in supernatants was determined by ELISA (Cytometric BeadsArray, Becton Dickinson, Heidelberg, Gennany). Data are shown asmean±SD. ** Results differed from Control (PBS) group (p<0.01;Mann-WhitneyU test; n=3-9 per group).

FIG. 17A: Alignment of nucleic acid sequences of EPO deletion variants;FIG. 17B: Alignment of nucleic acid sequences of EPO deletion variants(continued from FIG. 17A).

FIG. 18: DNA sequences of mutants and deletion variants createdrecombinantly as well as wild type Helix A (hWT-EPO Helix A). Herein SEQID NO: 55 is hA (Wild type Helix A), SEQ ID NO: 56 is hAmA (deletionmutatant with Alanin), SEQ ID NO: 57 is hAmE (deletion mutant withglutamic acid), SEQ ID NO: 58 is hA-10(deletion mutant Helix A minus 10aa) and SEQ ID NO: 59 is hA-20 (deletion mutant Helix A minus 20 aa).

FIG. 19A: A preferred embodiment wherein the leader transport sequencesare deleted is depicted in which the hA DNA sequence without leader asSEQ ID NO: 60. This is the mature exported protein. It also shows theleader-sequence (SEQ ID NO: 63). FIG. 19B shows hA amino acid withoutleader as SEQ ID NO: 61 and the amino acid sequence of theleader-sequence as SEQ ID NO: 62.

EXAMPLES

Synthesis of Murine EPO cDNA

RNA was isolated from kidneys of wild type C57BL/6 or SV129S6 mice orfrom two different mouse brains (1 hour after stroke) by trizolextraction. The RNA was precipitated with chloroform and isopropanol andfinally dissolved in DEPC-H₂O. DNA was digested to the RQ1 RNase-freeDNase protocol from Promega. The reaction was stopped by addition of 200μl phcnollchloroformlisopropyl alcohol (25/24/I) to the reaction mix andcentrifugation for 10 min at 10000 rpm and 10° C. The supernatant wasmixed with 200 μl chloroform/isopropyl alcohol (24/1) and centrifugedfor 10 min at 10000 rpm and 10° C. 20 μl 8 M lithium chloride and 550 μlabsolute ethanol were added to the supernatant This mix was thenincubated for 1 h at −70° C. and subsequently precipitated for 30 min bycentrifugation at 11000 rpm and 0° C. The resulting pellet was washedwith 600 μl 75% ethanol, centrifuged at 8000 rpm (4° C., 10 min) anddried at room temperature. The RNA was dissolved in 20 μl DEPC-H₂0.

Moloney murine leukemia virus reverse transcriptase (MuL V, RNase Hminus, purchased from Promega) was employed in first strand cDNAsynthesis in a 15 μl reaction volume with DEPC-H₂O comprising 3 μg RNAand 3 μl random hexamer primer (10 μM). Reverse transcription wascarried out with 6 μl M-MuLV reaction buffer (5×), 2 μl dNTP (2.5 1 nMeach), 1 μl RNase inhibitor (1 U/μl), 1 μl M-MuLV reverse transcriptaseand 5 μl DEPC-H₂O in a PCR machine running the following program: 5 minat 2° C; 1 h at 37° C.; 5 min at 95° C.

The resulting cDNA pool was used top amplify the complete EPO cDNA by aNested PCR approach. The first step employed primers lying outside ofthe coding region of the EPO gene (genepo_sense (SEQ ID NO 39) gaa cttcca agg atg aag act tgc agc and genepo_antisense; (SEQ ID NO 40); gtggca gca gca tgt cac ctg tc). The second step used primers designed toamplify the gene from start to stop codon, with attached BamHII cleavingsites for the subsequent cloning (epo_sense (SEQ ID NO 41 tat gga tccatg ggg gtg ccc gaa cgt ccc ac and epo_antisense (SEQ ID NO 42 tat ggatcc tca cct gtc ccc tct cct gca gac). All primers were from MWG-BiotechAG. A nested PCR was performed in a Hybaid PCR machine in two steps,first PCR (3 min at 95° C.: 35 cycles: 30 sec at 65° C., 1 min at 72°C.,30 sec 95° C.; 10 min at 72° C.; 4° C. hold) and second PCR (3 min at95° C.; 5 cycles: 30 sec at 67° C., 1 min at 72° C., 30 sec at 95° C.;15 cycles: 30 sec at 70° C., 1 min at 72° C., 30 sec at 95° C.; 10 minat 72° C.; 4° C.).

In both PCRs, Pfu Turbo Hotstart DNA Polymerase (Stratagene) was usedaccording to the manufacture's protocol. The PCR product of the firststep was diluted 1:50 for the second PCR. A second cDNA synthesisprotocol was performed using the Access RT-PCR System (Invitrogen) withthe following parameters: 48° C. 5 min; 94° C. 2 min; 40 cycles: 94° C.30 sec, 65° C. 1 min, 70° C. 2 min; 70° C. 7 min; 4° C. The second PCRwas performed as described above.

The amplified full-length EPO cDNA and the EPO isomers were separated ona 1.2% TAE-agarose gel. A picture of the various PCR produces is shownin FIG. 1a . The fragments were than purified using the Wizard SV-GelCleanup System (Promega) or the Gel Extraction Kit (Qiagen, Hilden,Germany). As Pfu Polymerase generates blunt end products, the cDNA wassubcloned in the pCR-Blunt II-TOPO Vector using chemically competentTop10 One Shot Cells from (both Invitrogen).

Plasmid-DNA was isolated out of single colonies by usage of the QiagenQLA prep Kit. Inserts were sequenced on an ALFepress™ DNA Sequencer(Pharmacia Biotech) using the Thermo Sequenase™ Primer Cycle SequencingKit (Amersham Biosciences). The primers M13WDCY (SEQ ID NO 43: gtc gtgact ggg aaa acc ctg gcg) and M13REVCY (SEQ ID NO 44 agc gga taa caa tttcac aca gga) were labelled with Cy5. The parameters for sequencing weret=900 min; T=55° C.; 800V; 55 mA and 30 W. The sequence analysisrevealed the existence of a novel variant of EPO lacking exon 4 andthree internally deleted variants. The nucleotide sequences are depictedin FIG. 2a and FIG. 2b and the encoded peptide sequences are depicted inFIG. 4. The nucleotide and peptide sequence of the EPO variant mScorresponds to SEQ ID NO 13 and SEQ ID NO 14, respectively. Thenucleotide and peptide sequence of the EPO variant mG3 corresponds toSEQ ID NO 15 and SEQ ID NO 16, respectively. the nucleotide and peptidesequence of the EPO variant mG5 corresponds to SEQ ID NO 17 and SEQ IDNO 18, respectively. The nucleotide and peptide sequence of the EPOvariant m301 corresponds to SEQ ID NO 19 and SEQ ID NO 20, respectively.The nucleotide and peptide sequence of the EPO variant mK3 correspondsto SEQ ID NO 21 and SEQ ID NO 22, respectively.

Synthesis of Human EPO cDNA

Human adult kidney (male) and fetal brain (male) poly A+ RNA waspurchased from Stratagene. cDNA was generated from 250 ng kidney RNA or200 ng brain RNA according to the Moloney murine leukaemia virus reversetranscriptase (MuLV, RNase H minus) as described above. The resultingcDNA pool was used to amplify the complete EPO cDNA using Pfu Polymerase(Stratagene) with the following primers: Hepo_sense (SEQ ID NO 45): gatggg ggt gca cga atg tcc tgc and Hepo_antisense (SEQ ID No 46): cac acctgg tca tct gtc ccc tgt c.

The PCR was performed in a PCR machine from Invitrogen (3 min at 95° C;35 cycles: 30 sec at 67° C., 1 min at 72° C., 30 sec at 95° C.; 10 minat 72° C.). In the case of the fetal brain cDNA a Nested PCR approachwas used, performing a second amplifying step on the PCR product of 20cycles. The amplified PCR products were separated on a 1.2% TAE-agarosegel (FIG. 1b ) and purified using the Gel Extraction Kit (Qiagen,Hilden, Germany). The purified cDNA was subcloned in the pCR-BluntII-TOPO Vector using chemically competent Top10 One Shot Cells (bothfrom Invitrogen). Plasmid-DNA was isolated out of single colonies byusage of the QIA prep kit (Qiagen, Hilden, Germany). Inserts waresequenced on an ALFexpress™ DNA sequencer (Pharmacia Biotech) using theThermo Sequenase™ Primer Cycle Sequencing Kit (Amersham Biosciences).The primers M13FWDCY (SEQ ID NO 43) and M13REVCY (SEQ ID NO 44) werelabelled with Cy5. The parameters for sequencing were: t=900 min; T=55°C.; 800 V; 55 mA and 30 W. The sequence analysis revealed the existenceof two novel variants of human EPO missing exon 3 and the first half ofexon 4, respectively, and a number of variants that follows the rule ofrepeated trimers or hexamers as detected in the mouse. The nucleotidesequences are depicted in FIG. 3a and FIG. 3b and the encoded peptidesequences are depicted in FIG. 4. The nucleotide and peptide sequence ofthe EPO variant hS3 corresponds to SEQ ID NO 1 and SEQ ID NO 2,respectively. the nucleotide and peptide sequence of the EPO varianth1-4 corresponds to SEQ ID NO 3 and SEQ ID NO 4, respectively. Thenucleotide and peptide sequence of the EPO variant h1-5 corresponds toSEQ ID NO 5 and SEQ ID NO 6, respectively. The nucleotide and peptidesequence of the EPO variant hS4 corresponds to SEQ ID NO 7 and SEQ ID NO8, respectively. The nucleotide and peptide sequence of the EPO varianth1-1 corresponds to SEQ ID NO 9 and SEQ ID NO 10, respectively. Thenucleotide and peptide sequence of the EPO variant h2-1 corresponds toSEQ ID NO 11 and SEQ ID NO 12, respectively.

Expression of His-Tagged Proteins in HEK Cells

BamHI and EcoRI restriction sites for cloning were added to both themouse and the human EPO variants by using overhang sense primers andoverhang antisense primers without stop codon (for mouse variants:epo_sense (SEQ ID NO 41) and epoeco_antisense (SEQ ID NO 47): aaa gaattc cct gtc ccc tct cct gca gac ctc; for human variants; hepobam_se (SEQID NO 48): tat gga tcc atg ggg gtg cac gaa tgt cc, heptoeco_as [SEQ IDNO 49]: aga gaa ttc tct gtc ccc tgt cct gca g). The PCR products werecloned into pcDNA-3.1-HIS/V5 A (Invitrogen) using BamHI and EcoRIrestriction sites. Plasmids were amplified in XL-1 Blue Competent Cells(recAI endAI gyrA96 thi1 hsdR17 supE44 relAI lac [F⁺ proAB lac1^(q)ZΔM15Tn10 (Tet^(R))]) (Stratagene). The XL-1 Blue Competent Cellstransformation protocol was performed without β-mercaptoethanol and witha prolonged heat pulse of 60 seconds. Plasmid DNA was extracted usingthe QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). Fortransfection into mammalian cells DNA was extracted using the EndoFreePlasmid Maxi Kit (Qiagen, Hilden, Germany). HEK 293 cells (BDbiosciences) were grown for 18 days in Dulbecco's modified Eagle'smedium (DMEM; Biochrom, Berlin, 1 g/l glucose, 3.7 g/l NaHCO₃;supplemented with 10% fetal calf serum GOLD, 1% penicillin/streptavidineand 1% L-glutamine) in tissue culture flasks (25 cm²) at 37° C. and 5%CO₂. Cells were split every 2-3 days after reaching 80-90% confluence.At DIV18 120,000 cells were plated per well in 12 well plate containingDulbecco's modified Eagle's medium without antibiotics. Cells were grownfor approximately 48 h till 50% confluence. Transfection was performedwith Lipofectamine 2000 (Invitrogen) adapting the provided protocol forHEK cells.

Plating medium of HEK-cells was replaced 10 min before transfection byserum-free DMEM without antibiotics. Cells were incubated 5 h at 37° C.with DNA-Lipofectamine complexes. Medium was then changed to freshserum-containing DMEM without antibiotics. At DIV2 cells were split andplated in Dulbecco's modified Eagle's with antibiotics.

Expression and Purification of His-tagged EPO Variants

His-tagged proteins were transiently expressed in HEK-cells. Medium fromHEK293 cells was harvested 2-6 days after transfection withpcDNA-3.1-HIS/V5 A—constructs. Cells debris was pelleted at 3500 rpm, 4°C. for 15 min. BD TALON™ Metal Affinity Resin (BD Biosciences) was usedfor purification of his-tag proteins. All steps (equilibration, washingand elution) were performed at pH 7.1. The provided protocol wasmodified to a prolonged overnight binding step at 4° C. Eluate wascollected in 500 μl-fractions. Fractions were analysed by Western Blotsusing an anti-rhEPO antibody from Santa-Cruz or a murine EPO ELISA-Kit(R&D). Imidazole was removed from protein-containing fractions usingHiTrap™ Desalting columns (5 ml) from Amersham Biosciences according tothe manufactures protocol. This included a change of buffer to PBS.

Western Blot

A 16% SDS-Gel was prepared using standard-protocols and run at 110 V.Blotting was done on nitrocellulose-membranes for 45 min at 200 mA. Theblot was blocked for at least one hour in blocking buffer containing 5%non-fat dry milk powder in 0.1% Tween-20. Incubation with the firstantibody (EPO (H-162) sc-7956 rabbit polyclonal IgG, Santa Cruz, 1:500)was performed over-night at 4° C. The secondary antibody (goatanti-rabbit HRP; 1:1000) was added for 2 hours at room-temperature. Theblot was revealed by use of Luminol; photos were exposed for 2 minutes.Membranes were stained with Ponceau Red. The EPO specific antibody wascapable of detection all EPO variants.

Erythroid Colony Formation Assay

Bone marrow cells were harvested from tibia and femur of male C57BL/6mice (8-11 weeks) and resuspended in α-medium (supplemented with 10%fetal calf serum GOLD, 1% penicillium/streptavidine and 1% L-glutamine).Cells were seeded in 35 mm² Petri dishes (225,000 cells/dish) containing8 parts Metho Cult SF 3236 methyl cellulose (StemCell Technologie Inc),1 part cells and 2 parts α-medium mixed with HEK-cell preconditionedmedium containing the EPO derivates (150 U/l in the case of murine EPO).150 U/l of rhEPO (Roche) was used as positive control. Plates wereincubated at 37° C. in a humidified atmosphere containing 5% CO₂ for 48hours. For evaluation only reddish colonies containing at least 6hemoglobinised cells were taken into account.

Hematopoietic Potential of the EPO Variants

Metho Cult SF 3236 triggers the formation of colonies (CFU-M, CFU-G orCFU-E) only after addition of the appropriate cytokines. Formation ofCFU-E (Colony forming unit-erythroblast) can be observed, after additionof erythropoietin, after 2 days. The small irregular reddish coloniesdisappear by day 3.

In this assay, the hematopoietic potential of the variants was testedand compared to wild type form of EPO as well as rhEPO. The followingconditions were prepared for comparison: medium from HEK cellstransfected with pZ/EG ns negative control, medium from HEK cellstranfected with pZ/EG cells plus 150U/I rhEPO (Roche) as a positivecontrol, and medium from HEK cells transfected with eitherpZ/EG-EPO-IRES (150U/I murine EPO), pZ/EG-Splice-IRES (variant S; mS) orpZ/EG-G3-IRES (variant G3; mG3). At DIV2 only reddish colonies werecounted containing at least 6 hemoglobinised cells. The results of threeindependent experiments are depicted in FIG. 5.

In comparison to murine EPO and rhEPO the murine EPO variants (mS andmG3-variant) lacked haematopoietic potential.

Primary Neuronal Cultures

Rat primary neuronal cultures were obtained from E16 to early E19embryos of Wistar rats (Bundesinstitut für gesundheitlichenVerbraucherschutz und Veterinärmedizin, Berlin, Germany). Cre-expressingmouse neurons were obtained from E16 embryos of heterozygous transgenicmice expressing Cre-recombinase under the control of the tubulin α-1promotor (provided by Dr. U, Schweitzer; Experimental Endocrinology,Charité). Murine and rat cultures were prepared according to a modifiedprotocol from Brewer (1995) J Neurosci Res. 42: 674-83. Cerebral cortexwas isolated after removal of meninges aad rinsed twice in PBS(Biochrom, Berlin, Germany). After 15 man incubation in trypsin/EDTA(0.05/0.02% w/v in PBS) at 37° C. tissues were rinsed twice in N-Med(modified Eagle's medium from Gibco with 10% fetal calf serum, 100 Upenicillin plus streptomycin from Biochrom, 2 mM L-glutamine, 100 IEinsulin/l, 10 mM HEPES and 44 mM glucose) and dissociated carefully in asmall volume of N-Med using a Pasteur pipette. Cells were pelleted atroom temperature by 2 min centrifugation at 210 g and resuspended in NBMstarter medium (Neurobasal medium from Gibco with 2% B27 supplement fromGibco, 1% Pen/Strep, 0.5 mM L-glutamine and 25 μM glutamate).

Preparation of Culture Plates

24-well plates and 6-well plates were pretreated by over-nightincubation at 4° C. with poly-L-lysin from Biochrom (2.5 μg/ml in PBS).Rinsing of the wells with PBS was followed by 1 h incubation at 37° C.with coating medium (modified Eagles's medium with 5% FCS Gold from PAA,1% Pen/Strep, 10 mM HEPES and 0.03 w/v collagen G from Biochrom), thenthe wells were carefully rinced twice with PBS. Volume and type ofplating medium was chosen depending on experimental procedure.

Oxygen Glucose Deprivation in Rat Primary Cortical Neurons—A CellCulture Model of Cerebral Ischemia

For OGD the culture medium was washed out by rinsing once with PBS. OGDwas induced with 500 μl of a deoxygenated aglycemic solution (BSS₀-O₂;143.8 mM Na⁺, 5.5 ; mM K⁺, 1.8 mM Ca²⁺, 0.8 mM Mg²⁺, 125.3 mM Cl⁻, 26.2mM HCO₃ ⁻ and 0.8 mM SO₄ ²⁻, pH 7.4) in a hypoxic atmosphere generatedby a dedicated, humidified gas-tight incubator (Concept 400, RuskinnTechnologies, Bridgend, UK) flushed with a gas mix containing 5% CO₂,85% N₂ and 10% H₂. OGD-time depended on the density and the age of theculture and varied between 2 h 30 min and 2 h 40 min. In controlexperiments the wells were treated with 500 μl of the oxygenatedglycemic BSS₀ solution (BSS₀+O₂; 143.8 mM Na⁺, 5.5 mM K⁺, 1.8 mM Ca²⁺,0.8 mM Mg²⁺, 125.3 mM Cl⁻, 26.2 mM HCO₃ ⁻, 0.8 mM SO₄ ²⁻, and 20 mMglucose, pH 7.4) and incubated at 37° C. in a normoxic atmospherecontaining 5% CO₂. Immediately after OGD, treated cells and controlswere changed from BSS solution to 500 μl of medium containing 40%conditioned NBM plus 60% fresh NBM. After 24 h, lactate dehydrogenase(LDH) activity was measured in the supernatants as an indicator of celldeath.

For LDH measurement 25 82 l of medium was mixed with 100 μl fresh β-NADHsolution (0.15 mg/ml in 1×LDH-buffer; Sigma, reduced form) in a 96 wellsplate (Greiner). 25 μl of 22.7 mM pyruvate (Sigma) was added immediatelybefore placing the plate into the Reader (Thermo Labsystems; MRX^(TC)Revelation). Parameters were chosen as follows; filter: 340 nm, shaketime: 5 sec, interval: 30 sec, counts: 10. LDH-concentration wascalculated proportionally to the LDH-standard (Greiner, systemcalibrator).

Induction of Neuroprotection by Conditioned Medium from TransfectedHEK293 Cells Expressing EPO Variants

In the following experiments rhEPO (recombinant human EPO from SigmaAldrich, Deisenhofen, Germany) was used as a positive control.Neuroprotection assays are schematically depicted in FIG. 6. Neuronswere plated in 24-well plates at a density of 300,000 cells in a finalvolume of 600 μl NBM starter medium. After 4 days, 200 μl of the mediumwas replaced by 250 μl fresh NBM (same as NBM starter withoutglutamate).

For pretreatment with rhEPO, wild type mEPO, wild type hEPO or EPOvariants the medium was removed to an end volume of 200 μl and filled upwith 200 μl fresh NBM+B27 containing equimolar amounts (corresponding to200 U/l rhEPO) of EPO or EPO variants, respectively. Equivalentconcentrations of the various EPO variants (as well as mEPO and hEPO) inthe conditioned medium from HEK293 cells were estimated by Western blotand EPO-Elisa. Thereafter neurons were grown for 48 h under normoxic,humified conditions at 37° C. before oxygen glucose deprivation (OGD)was performed (OGD interval as indicated). Cell death was assessed 24 hafter OGD by measurement of LDH release. Reduction in LDH release,compared to mock-treated neurons (medium from HEK293 cells transfectedwith the backbone plasmid; =ko; 100%), is a quantitative measure ofneuroprotection. In all experiments we observed a more robustneuroprotective effect provided by the EPO variants, if compared to wtEPO (see FIG. 7 Panel A and B for murine EPO and variants thereof andFIG. 8 Panel A and B for human EPO and variants thereof).

The neuroprotection induced by the murine EPO variants is more robustthan that induced by EPO (rhEPO as well as wild type mouse EPO). Forexample, neuroprotection mediated by EPO can only be observed in aclearly defined window of OGD length (corresponding to a clearly defineddamage level). At low concentration the neuroprotection by hS3 and hS4was equal or better than the neuroprotection of wt hEPO. Overall,neuroprotection induced by the variants is stronger than that induced byrhEPO. In addition, variants have an higher neuroprotective potentialthan both wild type forms mEPO and hEPO, which were produced by the sameprocedure as the EPO variants.

H9c2—Model of Ischemia

The rat BDIX heart myoblast cell line (obtained from European Collectionof Cell Cultures) was cultured in DMEM (Biochrom) containing 4.5 g/lglucose supplemented with 2 mM L-glutamine, 10% inactivated fetal calfserum and 1% penicillin-streptavidin. Subconfluent cultures (70%) weresubcultured 1:4. Cells were plated in 400 μl medium containing 120 pMhePO or hS3 respectively in a density of 15,000 cells per well in24-well plates and cultured for 48 hours. Hypoxia was achieved byculturing the cells in 400 μl serum-deficient DMEM containing 4.5 g/lglucose supplemented with 2 mM L-glutamine and 1%penicillin-streptavidin and leaving them for 24 h in an anaerobicworkstation (Concept 400, Ruskinn Technologies, Bridgend, UK) saturatedwith a gas mix containing 5% CO₂, 85% N₂ and 10% H₂ at 37° C. Controlcells were left in serum-deficient DMEM in a normoxic incubator. At theend of the experiment medium was replaced to 400 μl freshserum-deficient DMEM and LDH was measured according to standardprotocols 24 h later.

Immunoprecipitation

Male 129S6 mice or male C57BI6 mice (8-10 weeks, Bundesinstituts fürRisikobewertung, Berlin) having free access to food and water were usedfor the experiments. CoCl2 was injected subcutanely in a dose of 60mg/kg and animals were killed 18 hours later. Protein expression wasmeasured in serum, kidney and brain protein extracts by a commercialavailable ELISA (R&D), mEPO).

Antibodies for immunoprecipitation were purchased from R&D (anti-mEPOantibody, goat, biotin-labelled) and Santa-Cruz (anti-rhEPO, rabbit).Immunoprecipitation was performed according to standard protocols andevaluated by western blot.

Blocking of the western blot detection antibody was achieved by twohours incubation with 10 μg DarbpoietinA at room temperature prior toblot incubation.

Generation of Alpha-Helix-Mutants (FIG. 10)

Human alpha-helix-mutants were all generated by PCR based approachesusing standard protocols.

Mutant A (hAmA) and mutant E (hAmE) are variants of the alpha-helix withamino acid exchange at position 41 (arginine). cDNA sequence was changedfrom AGG to GCG for mutant A (alanine) or to GAG for mutant E(glutamate). −20aa and −10aa are deletion variants of the alpha-helixmissing 20 amino acids or 10 amino acids respectively at the c-terminus.All mutants were generated without V5 and His-tag and expressed in HEK293 cells. Neuroprotection experiments were performed as describedpreviously using medium of transfected HEK cells expressing thedifferent variants.

hEPO and HS3 Mediated Cytoprotection in a Model of Ischemia in H9-c2Cells (FIG. 11)

The cytoprotective potential of the EPO variants was shown exemplarilyfor purified hEPO and hS3 in a model of ischemia consisting of serumdeprivation and hypoxia in H9c2 cardiac myoblasts (FIG. 1). LDH releasewas assessed as a marker of apoptotic cell death. We found significantcytoprotective capacities for both variants (approximately 20% and 25%for hEPO and hS3).

Immunoprecipitation Reveals EPO Splicing Isoform in Kidney ProteinExtracts of CoCl2-treated Mice (FIG. 12)

To strengthen our finding of EPO splicing isoforms in human and murinetissues by a PCR-based approach we performed immunoprecipitations onmurine serum, brain and kidney protein extracts of CoCl2 treated miceusing antibodies tested to recognize both isoforms. Subcutane injectionof CoCl2 is known to increase erythropoietin levels in several mousetissues, namely blood, brain, liver and kidney.

We were able to precipitate erythropoietin (approximately 40 kDa) fromserum, brain sod kidney protein extracts of CoCl2 treated mice (FIG. 2);precipitation of erythropoietin from a kidney protein extract of anuntreated mouse failed due to the low expression level. Furthermore wewere able to prove the existence of a second smaller protein(approximately 30 kDa) in the kidney protein extract of CoCl2 treatedmice. This protein is specifically recognized by the anti-rhEPO antibodyas shown by complete blocking of the antibody-antigene interaction withDarbpoietin A. These findings strongly support tbe existence of a murineerythropoietin splicing isoform. These results were reproduced in asecond mouse strain, namely C57BI6.

Neuroprotection Mediated by Different Isoforms of the ErythropoietinAlpha-helix (FIG. 13)

Analysing the neuroprotective potentials of the so far identifiederythropoietin variants we suggested the alpha helix to be tbefunctionally important domain for the neuroprotective character oferythropoietin. In order to test this hypothesis we expressed ashortened form of human erythropoietin, namely the alpha-helix domain,in HEK 293 cells and tested this peptide in our OGD-model. We found anequivalent protective, potential with 30 pM and 15 pM of this peptide to30 pM of hEPO as shown in FIG. 13.

In order to identity the functional important residues in the alphahelix domains of human erythropoietin we generated differenterythropoietin mutants containing either amino acid exchanges (hAmA andhAmE) or complete domain deletions (hA-10 and hA-20).

Neither the neutral nor the acidic amino acid exchange at position 41was able to destroy the neuroprotective potential of the alpha-helix inour OGD model (FIG. 1).

Neuroprotection mediated by severed human EPO-isoforms (n=6) P*<0.05;ANOVA1 versus control (FIG. 14)

Deletion variants missing 10 or 20 amino acids at the c-terminus of thealpha-helix were expressed in HEK293 cells and also tested in theOGD-model. The deletion variant hA-10 had still neuroprotectiveproperties comparable to the hS3 splice isoform. Deletion of 20 aminoacids (hA-20) led to a peptide that was not protective anymore (FIG.15).

Immunomodulation by Human Erythropoietin Variants (FIG. 16)

The human EPO variants hS3 and hS4 exhibit strong immunomodulatoryeffects. In human macrophages stimulated with the endotoxinlipopolysaccharide (LPS) hS3 and hS4 induce the anti-inflammatorycytokine IL-10 and reduce the expression of the pro-inflammatorycytokines IL-6 aad IL-8. Compared to EPO (hWT) anti-inflammatory effectsof hS3 and hS4 are much more pronounced. These anti-inflammatoryproperties of EPO variants are useful in treatment of inflammatory (e.g.Multiple Sclerosis, viral and bacterial infections, sepsis) anddegenerative diseases (e.g. stroke, myocardial infarctions).

What is claimed is:
 1. An erythropoietin (EPO) variant encodingpolynucleotide or the complementary strand thereof, wherein thepolynucleotide is selected from the group consisting of: (a)polynucleotides encoding at least the mature form of the polypeptidestermed hs3, h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3, mG5, m301, mK3, ha,hAma, hAmE, hA-10 and ha-sequence without leader having the deducedamino acid sequence as shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16,18, 20, and 22, 50, 51, 52, 53 and 61 respectively; (b) polynucleotideshaving the coding sequence, as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 55, 56, 57, 58 and 60 encoding at least the matureform of the polypeptide; (c) polynucleotide encoding a humanized versionof the polypeptides mS, mG3, mG5, m301 and mK3 having the deduced aminoacid sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22, (d)polynucleotides encoding a polypeptide comprising an amino acid sequenceselected from the group of amino acid sequences as shown in SEQ ID NO24, 26, 28, and 30 fused to the N-terminus of an amino acid sequenceselected from the group of amino acid sequences as shown in SEQ ID NO32, 34, 36, and 38; (e) polynucleotides comprising a polynucleotidesequence selected from the group of polynucleotide sequences as shown inSEQ ID NO 23, 25, 27, and 29 fused to 5′ of a polynucleotide sequenceselected from the group of polynucleotide sequences as shown in SEQ IDNO 31, 33, 35, and 37; (f) polynucleotides encoding a derivative of apolypeptide encoded by a polynucleotide of any one of (a) to (e),wherein in said derivative between 1 and 10 amino acid residues areconservatively substituted compared to said polypeptide, and saidderivative comprises APPRLICDSRVLERYL (SEQ ID NO: 79) orAPPRLICDSRVLERYI (SEQ ID NO: 80); (g) polynucleotides encoding afragment of a polypeptide encoded by a polynucleotide of any one of (a)to (f), wherein in said fragment between 1 and 10 amino acid residuesare N- and/or C-terminally deleted, and said fragment comprisesAPPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80);(h) polynucleotides which are at least 95 % identical to apolynucleotide as defined in any one of (a) to (e) and which code forpolypeptide comprising APPRLICDSRVLERYL (SEQ ID NO: 79) orAPPRLICDSRVLERYI (SEQ ID NO: 80); and (i) polynucleotides encoding anEPO variant polypeptide, which comprises the N-terminal part of fulllength EPO including helix A comprising APPRLICDSRVLERYL (SEQ ID NO: 79)or APPRLICDSRVLERYI (SEQ ID NO: 80) and which lacks at least one of thefollowing: (1) a fragment of at least 20 amino acids between helix C andD; or (2) a fragment of at least 10 amino acids of helix D; (j)polynucleotides encoding a derivative of a polypeptide encoded by apolynucleotide of any one of (i), wherein in said derivative between 1and 10 amino acid residues are conservatively substituted compared tosaid polypeptide, and said derivative comprises APPRLICDSRVLERYL (SEQ IDNO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80).
 2. The polynucleotide ofclaim 1 which is DNA, genomic DNA or RNA.
 3. A vector containing thepolynucleotide of claim
 1. 4. The vector of claim 3 in which thepolynucleotide is operatively linked to expression control sequencesallowing expression in prokaryotic and/or eukaryotic host cells.
 5. Amethod for the treatment or prevention of a condition associated withtissue damage due to cell death comprising using a polypeptide havingthe amino acid sequence encoded by a polynucleotide, wherein thepolynucleotide is selected from the group consisting of: (a)polynucleotides encoding at least the mature form of the polypeptidestermed hs3, h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3, mG5, m301, mK3, ha,hAma, hAmE, hA-10 and ha-sequence without leader having the deducedamino acid sequence as shown in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16,18, 20, and 22, 50, 51, 52, 53 and 61 respectively; (b) polynucleotideshaving the coding sequence, as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 55, 56, 57, 58 and 60 encoding at least the matureform of the polypeptide; (c) polynucleotide encoding a humanized versionof the polypeptides mS, mG3, mG5, m301 and mK3 having the deduced aminoacid sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22, (d)polynucleotides encoding a polypeptide comprising an amino acid sequenceselected from the group of amino acid sequences as shown in SEQ ID NO24, 26, 28, and 30 fused to the N-terminus of an amino acid sequenceselected from the group of amino acid sequences as shown in SEQ ID NO32, 34, 36, and 38; (e) polynucleotides comprising a polynucleotidesequence selected from the group of polynucleotide sequences as shown inSEQ ID NO 23, 25, 27, and 29 fused to 5′ of a polynucleotide sequenceselected from the group of polynucleotide sequences as shown in SEQ IDNO 31, 33, 35, and 37; (f) polynucleotides encoding a derivative of apolypeptide encoded by a polynucleotide of any one of (a) to (e),wherein in said derivative between 1 and 10 amino acid residues areconservatively substituted compared to said polypeptide, and saidderivative comprises APPRLICDSRVLERYL (SEQ ID NO: 79) orAPPRLICDSRVLERYI (SEQ ID NO: 80); (g) polynucleotides encoding afragment of a polypeptide encoded by a polynucleotide of any one of (a)to (f), wherein in said fragment between 1 and 10 amino acid residuesare N- and/or C-terminally deleted, and said fragment comprisesAPPRLICDSRVLERYL (SEQ ID NO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80);(h) polynucleotides which are at least 95 % identical to apolynucleotide as defined in any one of (a) to (e) and which code forpolypeptide comprising APPRLICDSRVLERYL (SEQ ID NO: 79) orAPPRLICDSRVLERYI (SEQ ID NO: 80); and (i) polynucleotides encoding anEPO variant polypeptide, which comprises the N-terminal part of fulllength EPO including helix A comprising APPRLICDSRVLERYL (SEQ ID NO: 79)or APPRLICDSRVLERYI (SEQ ID NO: 80) and which lacks at least one of thefollowing: (1) a fragment of at least 20 amino acids between helix C andD; or (2) a fragment of at least 10 amino acids of helix D; (j)polynucleotides encoding a derivative of a polypeptide encoded by apolynucleotide of any one of (i), wherein in said derivative between 1and 10 amino acid residues are conservatively substituted compared tosaid polypeptide, and said derivative comprises APPRLICDSRVLERYL (SEQ IDNO: 79) or APPRLICDSRVLERYI (SEQ ID NO: 80).
 6. A method according toclaim 5, wherein said cell death is induced by ischemia, hypoxia,bacterial infection, viral infection, autoimmunologically,traumatically, chemically induced, or radiation induced.
 7. A methodaccording to claim 5, wherein said condition is an acute or chronicneurodegenerative and/or neuroinflammatory disorder, is an acute orchronic disorder of the heart, lung, kidney, liver or pancreas or saidcondition is associated with an organ or cell transplantation.
 8. Amethod according to claim 7, wherein said acute neurodegenerative and/orneuroinflammatory disorder is selected from the group consisting ofcerebral ischemia or infarction including embolic occlusion andthrombotic occlusion, reperfusion following acute ischemia, perinatalhypoxic-ischemic injury, cardiac arrest, intracranial hemorrhage,subarachnoidal hemorrhage and intracranial lesions, spinal cord lesions,intravertebral lesions, whiplash shaken infant syndrome, infectiousencephalitis, meningitis, headache.
 9. Method according to claim 7,wherein said chronic neurodegenerative and/or neuroinflammatory disorderis selected from the group consisting of dementias, Pick's disease,diffuse Lewy body disease, progressive supranuclear palsy(Steel-Richardson syndrome), multiple sclerosis, multiple systematrophy, chronic epileptic conditions associated with neurodegeneration,motor neuron diseases, degenerative ataxias, cortical basaldegeneration, ALS-Parkinson's Dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies, primary progressive aphasia, striatonigraldegeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 andolivopontocerebellar degenerations, Gilles De La Tourette's disease,bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy(Kennedy's disease), primary lateral sclerosis, familial spasticparaplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease,Tay-Sach's disease, Sandhoff disease, familial spastic disease, spasticparaparesis, progressive multifocal leukoencephalopathy, familialdysautonomia (Riley-Day syndrome), polyneuropathies, prion diseases,addiction, affective disorders, schizophrenic disorders, chronic fatiguesyndrome, chronic pain.
 10. A method according to claim 5, wherein saidcondition is aging.
 11. A method according to claim 5, wherein themedicament is administered prior to or after the onset of saidcondition.